FIELD OF THE INVENTION

The present invention relates to chromenone derivatives, to a process for preparing the chromenone derivatives, to a pharmaceutical composition comprising the chromenone derivatives and to the use of the chromenone derivatives in the treatment of neurodegenerative disease.

BACKGROUND OF THE INVENTION

Neurodegenerative diseases result from deterioration of neurons or their myelin sheath which over time will lead to dysfunction and disabilities resulting from this. Adult mammalian brain has limited capacity for regeneration. This makes the repair of any injuries hazardous and, consequently, CNS traumas are devastating. Regulation, or production of microglia by the immune system, in a process of neuroinflammation, is currently being rigorously studied for its role in neurodegenerative diseases.

These diseases are divided into two groups according to phenotypic effects, causing problems with movements, such as ataxia, or affecting memory and related to dementia. The most common neurodegenerative diseases are Alzheimer's, Parkinson's, multiple sclerosis, amyotrophic later sclerosis (ALS) and Huntington's. But neurodegeneration also happens as a result of a stroke, a blow to the spinal cord or head, or bleeding in the brain.

Alzheimer's disease (AD) is the most common cause of dementia that gradually destroys neurons and affects more than 24 million people worldwide. It occurs mostly in older adults and patients afflicted with AD lose their ability to learn, remember, make decisions, communicate and carry out daily activities. The etiology and progression of AD is not well understood, but is associated with amyloid beta (AÎ²) plaques and neurofibrillary tangles in the brain.

Parkinson's disease (PD) is a degenerative disorder of the central nervous system affecting more than 6 million people worldwide and that often impairs the sufferer's motor skills and speech. The symptoms of Parkinson's disease result from the loss of dopamine-secreting cells in the region of the substantia nigra (literally â€œblack substanceâ€ ). These neurons project to the striatum and their loss leads to alterations in the activity of the neural circuits within the basal ganglia that regulate movement.

Multiple sclerosis (MS) is a neurological disease of the young adult which associates demyelination with inflammatory or even immunological elements. For the majority of patients, MS is known to evolve initially by â€œrelapsing and remittingâ€ , and then in a â€œprogressiveâ€ form affecting more than 2.5 million people worldwide. The most common initial symptoms reported are: changes in sensation in the arms, legs or face, complete or partial vision loss, weakness, unsteadiness when walking, and balance problems.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that results from the death of motor neurons. A progressive loss of muscle control impairs the individual's capacity for independent function. ALS strikes the cells in the brain and spinal cord (motor neurons), which send signals to move muscles. In some cases, a gene causes a mutation in a protein (called SOD1) that normally â€œcleansâ€ up toxic particles inside a cell. When SOD1 is mutated, toxic particles accumulate inside motor neurons causing them to malfunction. But this mutation only explains a few percent of cases of ALS. The cause of ALS, which afflicts about 350,000 adults worldwide, is unknown.

Huntington's disease (HD) is a fatal hereditary disease with profound neurological and behavioral features. HD is typically characterized by uncontrollable movements and psychological disturbances. It is caused by a detectable mutation in the gene coding for a protein known as huntingtin. The normal huntingtin protein is present in all brain cells and is required for cell survival. However, when the gene is mutated, the resulting protein is altered and behaves differently, making some brain cells vulnerable. Affected areas include the cortex (which controls planning, thought, and memory) and the basal ganglia (which coordinates movement). Currently, there are no treatments for HD or ability to slow its progression.

Stroke and traumatic brain injury can also cause neuronal loss and lead to cognitive decline. Stroke can be classified into two major categories: ischemic and hemorrhagic. Ischemia is due to interruption of the blood supply, while hemorrhage is due to rupture of a blood vessel or an abnormal vascular structure. Stroke can cause permanent neurological damage, complications and death if not promptly diagnosed and treated. It is the third leading cause of death and the leading cause of adult disability in the United States and Europe.

Prions diseases are fatal neurodegenerative diseases caused by an agent known as a â€œprionâ€ . The disorders cause impairment of brain function, including memory changes, personality changes and problems with movement that worsen over time. Prion diseases of humans include classic Creutzfeldt-Jakob disease, Gerstmann-StrÃ¤ussler-Scheinker syndrome, fatal familial insomnia and kuru. The degenerative tissue damage caused by human prion diseases is characterized by four features: spongiform change, neuronal loss, astrocytosis and amyloid plaque formation.

Frontotemporal Dementia (FTD) accounts for 18% of dementias in people under 65 years old. It frequently manifests itself as a behavioral disturbance, and can progress to impair an individual's capacity for independent thought and function. Recent studies have uncovered genetic factors that contribute to this dementia; however no treatment yet exists to block the brain deterioration it causes.

At present, most treatments of these incurable neurological disorders are symptomatic. The discovery of the presence of neural stem/progenitor cells (NSCs) in the adult brain has opened new approaches for therapeutic interventions [Martino G et al., Nat Rev Neurosci. 2006, 7, 395]. NSCs are multipotent progenitor cells which can differentiate into all different nervous cell types (e.g. neurons, astrocytes and oligodendrocytes) and can contribute to neurogenesis in adulthood. Neurogenesis is known to persist throughout adulthood in two regions of the mammalian brain: the subventricular zone (SVZ) of the lateral ventricles and the dentate gyrus of the hippocampus. In these regions, NSCs continue to divide and give rise to new functional neurons and glial cells [Zhao C et al., Cell. 2008, 132, 645]. It has been shown that a variety of factors can stimulate adult hippocampal neurogenesis (e.g. adrenalectomy, voluntary exercise, enriched environment, hippocampus dependent learning and anti-depressants).

The differentiation of these NSCs is regulated by key factors involved in neurogenesis and stem cell development. Hence, potent inducing agents for NSCs differentiation represent an innovative approach with good potential to treat or even cure neurological diseases by resupplying the central nervous system (CNS) with new functional nerve cells. Some protein growth factors (e.g., fibroblast growth factor (FGF) and epidermal growth factor (EGF)) are able to induce the differentiation of NSCs into mature neural cells [Caldwell M A et al., Nat. Biotechnol. 2001, 19, 475]. However, few non-protein synthetic molecules have been shown to confer such effect. [Coowar D et al., J Med. Chem. 2004, 47, 6270][Warashina M et al., Angew Chem Int Ed. 2006, 45, 591] [Saxe J P et al., Chem. Biol. 2007, 14, 1019].

Notch signaling controls a wide variety of cell fate decisions, including the differentiation of NSCs during neurogenesis [Artavanis-Tsakonas, S, Science. 1999, 284, 770]. Hence, compounds inhibiting the Notch signaling pathway would increase the production of neurons from NSCs. Inhibition of Notch signaling pathway represents a promising drug discovery tool for the treatment of different kinds of neurological diseases and disorders, such as Alzheimer disease (Beher D et al., Exp Opin Investigat Drugs. 2005, 14, 1385); multiple sclerosis [John G R et al., Nat. Med. 2002, 8, 1115]; brain tumours [Miele L et al., Curr Mol. Med. 2006, 6, 905], and autoimmune disorders [Briend E et al., Curr Opin Mol. Ther. 2005, 7, 56].

Although inflammation is not strictly a CNS disorder, it is frequently associated with brain injury, neurodegenerative diseases, and radiation treatment for brain tumors, which often causes deficits in cognition. Adult neurogenesis is also down-regulated by endotoxin-induced inflammation and can be restored by anti-inflammatory treatments [Ekdahl C T et al., PNAS. 2003, 100, 13632]. During the development of neurodegenerative or demyelinating diseases, microglial cells, the brain resident monocyte-macrophage cell population, become highly activated [Klegeris A et al., Curr Opin Neurol. 2007, 20, 351]. This activation produces large amounts of devastating pro-inflammatory cytokines, like tumour necrosis factor-Î± (TNF-Î±), interleukin-1Î² (IL-1Î²) as well as free radicals like nitric oxide (NO) and superoxide anion (O₂.^âˆ’). In AD, the extracellular depositions of amyloid beta (AÎ²) represent the major histological lesion and are responsible for the death of neurons by a currently unclear mechanism. One theory involves neuroinflammation which is supported by studies showing clustering of microglial cells within AÎ² depositions in human brain tissues. According to the neuroinflammatory hypothesis of neurodegenerative diseases, drugs with an anti-inflammatory mode of action should slow the disease progression.

The complex nature of the pathogenesis of neurodegenerative disease may require simultaneous use of several drugs directed at various factors contributing to the disease pathogenesis, including neuroinflammatory reactions. The urgent need for such combination therapies is being recognized due to the failure, or marginally protective effects, observed by using single anti-inflammatory and other neuroprotective agents. Hence, anti-inflammatory compounds promoting neurogenesis and/or the differentiation of oligodendrocyte precursors are excellent agents for the treatment of neurodegenerative or demyelinating diseases. Moreover, if these compounds modulate cell differentiation through inhibition of the Notch signaling pathway, they can be useful for the treatment of cancer where the Notch signaling is up-regulated.

Previous studies have shown that n-hexacosanol, a long chain primary alcohol containing 26 carbon atoms, confers neurotrophic activity. It has been shown that the length of the chain and the Ï‰-hydroxyl function are essential for the biological activity [Borg J et al., FEBS Lett. 1987, 213, 406]. Besides, in order to take into account the presumed contribution of inflammatory and oxidative phenomena in neurodegenerative pathologies, small molecules bearing a Ï‰-alkanol side chain combined with a neuroprotective moiety were developed. Structure-activity relationship studies led to the identification of compounds, presenting even better neurotrophic activities with a shorter chain length. Moreover, some of these fatty alcohol derivatives were able to induce differentiation of neural stem cells into mature neurons or to induce the differentiation of oligodendrocyte precursors into mature oligodendrocytes.

Tocopherol [WO2005030748A1] and Resveratrol [WO2007099162A2] long-chain alcohol compounds are known to have these dual bioactivies, promoting the differentiation of neural progenitor cells and modulating neuroinflammation. Both types of compound are known to act through the Notch signaling pathway.

WO2004007475A1 discloses certain flavonoids, including chromenone derivatives, bearing a C₂-C₃₀saturated or unsaturated hydrocarbon chain. The compounds are asserted to be useful as therapeutic antioxidants in the treatment of diseases or disorders involving oxidative damage. Neurological disorders, in particular Alzheimer's disease and Parkinson's disease are amongst the conditions that are described.

It has now been found that certain chromenone derivatives bearing a co-hydroxyalkyl chain promote differentiation of neural progenitor cells and modulate neuroinflammation. It will be appreciated that this technical effect is quite different from that described in WO2004007475A1. Applicant has tested an alkyl-substituted chromenone derivative of the kind described in WO2004007475A1 for the same effect, and found that it is active, but substantially less potent.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of an experiment to measure the ability of test compounds to down-regulate iNOS and TNF-Î±, gene expression in microglial cells.

FIG. 2 shows the results of an experiment to investigate the ability of test compounds to inhibit the Notch signaling pathway, by measuring Hes5 gene expression and Mash1 transcripts in the treated cultures.

FIG. 3 shows the results of an experiment to measure the ability of test compounds to induce the differentiation of SH-SY5Y neuroblastoma cell line by modulating the gene expression of Hash1 and Notch1.

DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides a compound of the general formula (I) or (II)

[Image Omitted]

or a pharmaceutically acceptable salt thereof, wherein each of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄and R₁₅independently represents a hydrogen atom, a hydroxyl group, a (C₁-C₆) alkyl group, a (C₂-C₆) alkenyl group, a (C₂-C₆) alkynyl group, a (C₁-C₆) alkoxy group, a hydroxy(C₁-C₆) alkyl group, a (C₁-C₆) alkoxy(C₁-C₆) alkyl group, a (C₁-C₆) alkanoyloxy group, a di(C₁-C₆) alkylaminoalkoxy group, a (C₁-C₆) alkanoyl group, a carboxyl group, a (C₁-C₆) alkoxycarbonyl group, a carbamoyl group, a (C₁-C₆) alkylaminocarbonyl group, a di(C₁-C₆) alkylaminocarbonyl group, a carbamoyloxy group, a (C₁-C₆) alkylaminocarbonyloxy group, a di(C₁-C₆) alkylaminocarbonyloxy group, a halogen atom, a halo(C₁-C₆) alkyl group, a halo(C₁-C₆) alkoxy group, a nitrile group, an amino group, a (C₁-C₆) alkylamino group, a di(C₁-C₆) alkylamino group or a (C₁-C₆) alkanoylamino group, a glucoside or two adjacent groups represent a methylenedioxy group, and n is an integer in the range of from 8 to 25.

Compounds according to the present invention can modulate the cellular fate of neural stem cells and promote the differentiation of these neural precursors to functional neurons and glial cells. In addition, compounds according to the present invention are able to reduce the inflammatory component of neurological disorders by modulating the activation of microglia and/or by reducing reactive gliosis.

The compounds are useful for the treatment of neurodegenerative diseases such as Alzheimer's disease, demyelinating diseases such as multiple sclerosis, Parkinson's disease, stroke (ischemic and hemorrhagic), traumatic brain injury and cancers, such as brain cancer.

Without wishing to be bound by theory, it is believed that the biological activity of these compounds is related to the inhibition of the Notch signaling pathway. Consequently, compounds according to the present invention are expected to be useful for the treatment of diseases associated with an up-regulated Notch signaling pathway activity, including cancer, in particular brain cancer.

In one embodiment, n in a compound of formula (I) is an integer in the range of from 10 to 20.

In another embodiment, n in a compound of formula (II) is an integer in the range of from 8 to 18.

Examples of particular values for n are 9, 10, 11, 12, 13 or 14 and 15.

In one embodiment, each of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄and R₁₅independently represents a hydrogen atom, a hydroxyl group, a (C₁-C₆) alkyl group, a (C₂-C₆) alkenyl group, a (C₂-C₆) alkynyl group, a (C₁-C₆) alkoxy group, a hydroxy(C₁-C₆) alkyl group, a (C₁-C₆) alkoxy(C₁-C₆) alkyl group, a (C₁-C₆) alkanoyloxy group, a (C₁-C₆) alkanoyl group, a carboxyl group, a (C₁-C₆) alkoxycarbonyl group, a carbamoyl group, a (C₁-C₆) alkylaminocarbonyl group, a di(C₁-C₆) alkylaminocarbonyl group, a halogen atom, a halo(C₁-C₆) alkyl group, a halo(C₁-C₆) alkoxy group, a nitrile group, an amino group, a (C₁-C₆) alkylamino group, a di(C₁-C₆) alkylamino group or a (C₁-C₆) alkanoylamino group, or two adjacent groups represent a methylenedioxy group.

In another embodiment, each of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄and R₁₅independently represents a hydrogen atom, a hydroxyl group, a (C₁-C₆) alkoxy group, a (C₁-C₆) alkanoyloxy group, a di(C₁-C₆) alkylaminoalkoxy group, a di(C₁-C₆) alkylaminocarbonyloxy group or a glucoside. For example, each of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄and R₁₅may independently represent a hydrogen atom, a hydroxyl group or a (C₁-C₆) alkoxy group. An example of a (C₁-C₆) alkoxy group is methoxy. An example of a (C₁-C₆) alkanoyloxy group is acetate. An example of a di(C₁-C₆) alkylaminoalkoxy group is dimethyaminoethoxy. An example of a di(C₁-C₆) alkylaminocarbonyloxy group is dimethylcarbamate

In one embodiment, each of R₄and R₁₁represents a hydrogen atom.

In yet another embodiment, each of R₇and R₁₄represents a hydroxyl group, a (C₁-C₆) alkoxy group, a (C₁-C₆) alkanoyloxy group, a di(C₁-C₆) alkylaminoalkoxy group, a di(C₁-C₆) alkylaminocarbonyloxy group or a glucoside. For example, each of R₇and R₁₄may represent a hydroxyl group or a (C₁-C₆) alkoxy group. An example of a (C₁-C₆) alkoxy group is methoxy. An example of a (C₁-C₆) alkanoyloxy group is acetate. An example of a di(C₁-C₆) alkylaminoalkoxy group is dimethyaminoethoxy. An example of a di(C₁-C₆) alkylaminocarbonyloxy group is dimethylcarbamate.

In another embodiment, each of R₅and R₁₂represents a hydrogen atom, a hydroxyl group or a (C₁-C₆) alkoxy group. Examples of values for R₅and R₁₂are a hydrogen atom and a methoxy group.

In another embodiment, each of R₆and R₁₃represents a hydrogen atom, a hydroxyl group, a (C₁-C₆) alkoxy group, a (C₁-C₆) alkanoyloxy group, a di(C₁-C₆) alkylaminoalkoxy group. For example, R₆and R₁₃may represent a hydrogen atom, a hydroxyl group or a (C₁-C₆) alkoxy group. Examples of values for R₆and R₁₃are a hydrogen atom, a methoxy group, an acetate or a dimethylaminoethoxy group.

In another embodiment, each of R₁and R₉represents a hydrogen atom, a hydroxyl group, a (C₁-C₆) alkoxy group, a (C₁-C₆) alkanoyloxy group, a di(C₁-C₆) alkylaminoalkoxy group, a di(C₁-C₆) alkylaminocarbonyloxy group or a glucoside. Thus, for example, each of R₁and R₉may represent a hydrogen atom, a hydroxyl group or a (C₁-C₆) alkoxy group. For example, each of R₁and R₉may represent a hydrogen atom, a hydroxy group, a methoxy group, an acetate, a dimethylaminoethoxy group, a dimethylcarbamate or a glucoside.

In another embodiment, each of R₂, R₃and R₁₀represents a hydrogen atom, a hydroxy group or a (C₁-C₆) alkoxy group. An example of a (C₁-C₆) alkoxy group is a methoxy group.

In another embodiment, each of R₈and R₁₅represents a hydrogen atom, a hydroxyl group, a (C₁-C₆) alkoxy group, a (C₁-C₆) alkanoyloxy group, a di(C₁-C₆) alkylaminoalkoxy group, a di(C₁-C₆) alkylaminocarbonyloxy group or a glucoside. For example, each of R₈and R₁₅may represent a hydrogen atom, a hydroxyl group or a (C₁-C₆) alkoxy group. An example of a (C₁-C₆) alkoxy group is methoxy. An example of a (C₁-C₆) alkanoyloxy group is acetate. An example of a di(C₁-C₆) alkylaminoalkoxy group is dimethyaminoethoxy. An example of a di(C₁-C₆) alkylaminocarbonyloxy group is dimethylcarbamate.

A particular group of compounds is that of formula (Iâ€²)

[Image Omitted]

and pharmaceutically acceptable salts thereof, wherein n represents 12 or 13 and R₁represents a hydroxyl group, a (C₁-C₆) alkoxy group, a (C₁-C₆) alkanoyloxy group, a di(C₁-C₆) alkylaminoalkoxy group, a di(C₁-C₆) alkylaminocarbonyloxy group or a glucoside. Compounds of formula (Iâ€²) have been found to possess particularly outstanding activity compared with other compounds of formula (I). Particular mention may be made of:

3-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-(12-hydroxydodecyl)-4H-chromen-4-one (compound A), 3-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-(13-hydroxytridecyl)-4H-chromen-4-one (Compound B), and pharmaceutically acceptable salts thereof. Particular mention may also be made of compounds of formula (Iâ€²) in which R₁represents a (C₁-C₆) alkanoyloxy group, a di(C₁-C₆) alkylaminoalkoxy group, a di(C₁-C₆) alkylaminocarbonyloxy group or a glucoside, for example those wherein R₁represents an acetate, a dimethylaminoethoxy group, a dimethylcarbamate or a glucoside. Examples of such compounds are 2-(4-hydroxy-3-methoxyphenyl)-6-(12-hydroxydodecyl)-4-oxo-4H-chromen-3-yl acetate; 2-(4-hydroxy-3-methoxyphenyl)-6-(13-hydroxytridecyl)-4-oxo-4H-chromen-3-yl acetate; 2-(4-hydroxy-3-methoxyphenyl)-6-(12-hydroxydodecyl)-4-oxo-4H-chromen-3-yl dimethylcarbamate; and 2-(4-hydroxy-3-methoxyphenyl)-6-(13-hydroxytridecyl)-4-oxo-4H-chromen-3-yl dimethylcarbamate, and pharmaceutically acceptable salts thereof. Such compounds are expected to demonstrate reduced or inhibited phase 1 metabolism of the flavonol moiety and/or ameliorated intestinal adsorption after oral administration, compared with the corresponding compounds in which R₁represents a hydroxyl group.

As used herein, the term (C₁-C₆) alkyl refers to an unbranched or branched alkyl group having from one to six carbon atoms. An example of a (C₁-C₆) alkyl group is methyl.

The term (C₂-C₆) alkenyl refers to an unbranched or branched alkenyl group having from two to six carbon atoms.

The term (C₂-C₆) alkynyl refers to an unbranched or branched alkynyl group having from two to six carbon atoms.

The term (C₁-C₆) alkoxy group refers to an unbranched or branched alkoxy group having from one to six carbon atoms. An example of a (C₁-C₆) alkoxy group is methoxy.

The term (C₁-C₆) alkanoyl group refers to an unbranched or branched alkanoyl group having from one to six carbon atoms. An example of a (C₁-C₆) alkanoyl group is ethanoyl.

The term halogen atom refers to fluorine, chlorine, bromine and iodine.

The terms halo(C₁-C₆) alkyl group and halo(C₁-C₆) alkoxy refer respectively to a (C₁-C₆) alkyl group and (C₁-C₆) alkoxy group in which one or more, for example one, two or three hydrogen atoms has been replaced with a halogen atom.

It will be appreciated that certain compounds of formula (I) and (II) may form pharmaceutically acceptable salts with pharmaceutically acceptable bases or acids. It will also be appreciated that certain compounds of formula (I) and (II) contain a chiral center and may therefore be prepared and isolated in a stereochemically pure form.

The compounds of the general Formula (I) can be obtained by methods such as the following:

Process A: R₂â• H; R₁â• OZ where Zâ• H, Ac, C(O)NMe₂, Glucose

[Image Omitted]

Compounds 4 of the present invention shown in Process A can be obtained by the following four steps. The flavonols 1 were synthesized from the 5â€² bromo-2â€²-hydrohyacetophenones. Aldol condensation with the commercial benzaldehydes gave the corresponding chalcones, and then Algar-Flynn-Oyamada (AFO) oxidation gave the bromoflavonols. The latter was then reacted with the corresponding anhydrides or halides in pyridine to give the bromoflavonols 1. These compounds then underwent a Sonogashira reaction with the different alkynes in the presence of tetrakis(triphenyphosphine) palladium to give the corresponding compounds 2. Catalytic hydrogenation in the presence of palladium on charcoal afforded the substituted compounds 3. Compounds 3 substituted with methoxy groups were then demethylated using boron tribromide in dichloromethane to give the hydroxyl groups. The corresponding flavonol 3-acetates, 3-carbamates or 3-glucosides are expected to demonstrate reduced or inhibited phase 1 metabolism of the flavonol moiety and ameliorated intestinal adsorption after oral administration.

Process B: R₁, R₂, R₃, R₆, R₇â• OH or OMe; R₄, R₅, R₈â• H

[Image Omitted]

Compounds 10 of the present invention shown in Process B can be obtained by the following six steps. Compound 5 is obtained through protection of commercial Quercetin with dimethylsulfate or benzyl bromide. The compound 5 was then iodinated selectively on position 5 using benzyltrimethyl ammonium chloroiodate (BTMA.ICl₂) and methylated or benzylated to give compound 7. The latter then underwent a Sonogashira reaction with the different alkynes to give the corresponding compounds 8. Catalytic hydrogenation in the presence of palladium on charcoal afforded the substituted compounds 9, which were demethylated using boron tribromide in dichloromethane to give the free hydroxyl groups.

Process C: R₂, R₃â• OH; R₁, R₄â• H

[Image Omitted]

Flavones long chain Ï‰-alkanols 20 of the present invention shown in Process C can be obtained by the following ten steps from commercial 2â€²,4â€²,6â€²-trihydroxyacetophenone. Following selective methylation of the latter compound, the flavones 14 were obtained in three steps using Baker-Vankataraman rearrangement. Selective demethylation with boron tribromide and iodination with benzyltrimethyl ammonium chloroiodate (BTMA.ICl₂) gave iodoflavones 17. These compounds then underwent a Sonogashira reaction with the different alkynes to give the corresponding compounds 18. Catalytic hydrogenation in the presence of palladium on charcoal afforded the substituted compounds 19, which were then demethylated using broron tribromide in dichloromethane to give the free-hydroxy flavones 20.

The compounds of the general Formula (II) can be obtained by methods such as the following:

Process D: R₉, R₁₀, R₁₃, R₁₄â• OH; R₁₁, R₁₂, R₁₅â• H

[Image Omitted]

Furo[2,3-f]flavonol long chain Ï‰-alkanols 23 of the present invention shown in Process D can be obtained in three steps from the iodomethylated quercetin 7. By modifying the Sonogashira reaction protocol i.e. using piperidine as solvent and base, we were able to devise a new single step method for the production of 9H-furo[2,3-f]benzopyran-9-one derivatives 21. These compounds then underwent catalytic hydrogenation and demethylation with boron tribromide to give compounds 23.

Process E: R₁₀â• OH; R₁₁â• H

[Image Omitted]

As in Process D, Furo[2,3-f]flavone long chain Ï‰-alkanols 27 of the present invention shown in Process E can be obtained in three steps from the iodomethylated flavones 24 using the Sonogashira-cyclisation tandem reaction. The compounds 27 are then obtained following catalytic hydrogenation and demethylation with boron tribromide.

According to another aspect, therefore, the present invention provides a process for the preparation of a compound of general formula (I) or (II) or a pharmaceutically acceptable salt thereof as defined hereinabove, which comprises

- a) for a compound of formula (II), reacting a compound of the general formula (III)

[Image Omitted]

or a protected derivative thereof, in which Z represents a leaving atom or group (for example a halogen atom such as a bromine atom or an iodine atom) with a compound of formula

â‰¡â€”(CH₂)_n-2â€”OP₁â€ƒâ€ƒ(IV)

in which P₁represents a hydrogen atom or a hydroxyl protecting group, in the presence of a Group VIII metal catalyst under cyclisation conditions (e.g. in the absence of a solvent);

- b) for a compound of formula (I), reducing a compound of the general formula (V)

[Image Omitted]

or a protected derivative thereof, in which P₂represents a hydrogen atom or a hydroxyl protecting group;

- followed by removing any protecting groups and if desired, forming a pharmaceutically acceptable salt.

The starting materials of formulae (V) and certain compounds of formula (III), which are useful for preparing compounds of formula (Iâ€²) are believed to be novel, and accordingly are provided as further aspects of the invention. The novel compounds of formula (III) may be represented by the formula (IIIâ€²)

[Image Omitted]

wherein Z represents a leaving atom or group (for example a halogen atom such as a bromine atom or an iodine atom) and R₁represents a hydroxyl group, a (C₁-C₆) alkoxy group, a (C₁-C₆) alkanoyloxy group, a di(C₁-C₆) alkylaminoalkoxy group, a di(C₁-C₆) alkylaminocarbonyloxy group or a glucoside; or salt thereof or a protected derivative thereof in which phenolic hydroxyl group and/or any hydroxyl group represented by R₁is or are protected with a protecting group, for example an aralkyl group such as a benzyl group.

Conveniently the compound and its pharmaceutically acceptable salts are administered to the patient in a pharmaceutical composition.

According to another aspect therefore, the present invention provides a pharmaceutical composition, which comprises a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

The compounds may be formulated in any conventional manner, for example in a tablet, capsule, suppository, solution, syrup or powder, depending upon the intended route of administration.

The dose of the compound administered to the patient will depend upon many different factors to be considered by the attending physician, including the age, weight and sex of the patient, the route of administration and the nature of the condition being treated. In general, the compound will be administered at a dose equivalent to administering the compound in the range of from about 0.01 mg/kg to about 100 mg/kg body weight.

In another aspect, the present invention provides a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, for use in therapy.

As described hereinabove, the compounds according to the present invention are useful in the treatment of neurodegenerative disease.

According to another aspect, therefore, the present invention provides a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, for use in the treatment of a neurodegenerative disease.

In another aspect, the present invention provides a method of treating a neurodegenerative disease in a patient requiring treatment, which comprises administering an effective amount of a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof as defined hereinabove.

The compounds according to the present invention may be administered alone or co-administered with compounds working by a different mechanism, for example neuroprotectant agents. In one embodiment, the compounds are co-administered compounds with an acetylcholinesterase inhibitor (e.g. Aricept) for Alzheimer's disease or L-DOPA for Parkinson disease]

The following examples illustrate the invention.

Example Synthesis of Compound 4 of General Formula (I)Example 1Synthesis of 6-bromo-2-(3,4-dimethoxyphenyl)-3-hydroxy-4H-chromen-4-one (1a)

To a solution of 3,4-dimethoxybenzaldehyde (2.0 g, 9.30 mmol, 1 eq) and 5â€²-bromo-2â€²-hydroxyacetophenone (1.55 g, 9.30 mmol, 1 eq) in dioxane (16 mL) and ethanol (23 mL), 40% (w/v) KOH solution (6.6 mL, 46.50 mmol, 5 eq) was added dropwise at 10Â° C. The reaction mixture was stirred for 144 h at room temperature. 150 mL CH₂Cl₂was added and the organic layer was washed with H₂O₂O (3*50 mL), dried over magnesium sulphate and concentrated in vacuo.

The yellow solid was dissolved in dioxane (34 mL) and ethanol (86 mL) and a 5.4% (w/v) NaOH solution (27.6, 37.50 mmol, 4 eq) was added dropwise. 30% H₂O₂solution (3.6 mL, 31.75 mmol, 3.4 eq) was added dropwise. The reaction mixture was stirred on ice for 2 h and subsequently for 24 h at room temperature, resulting in a yellow suspension. After acidification with 50 mL HCl (2 M), the aqueous phase was extracted with AcOEt (3*50 mL). The combined organic layers were washed with water, dried over magnesium sulphate and concentrated in vacuo. The product was recrystallized from ethanol to give 1.25 g (36%) of a yellow solid.

Molecular Weight: 377.19 (C₁₇H₁₃BrO₅).

¹H-NMR Î´ (TFA-d, 300 MHz) ppm (J in Hz): 3.99 (s, 3H, MeO), 4.00 (s, 3H, MeO), 7.12 (d, 1H, J=8.7, H-5â€²), 7.63 (d, 1H, J=9.0, H-8), 7.94 (dd, 1H, J=9.0, 2.1, H-7), 8.02 (d, 1H, J=2.0, H-2â€²), 8.08 (dd, 1H, J=8.7, 2.0, H-6â€²), 8.37 (d, 1H, J=2.1, H-5).

¹³C-NMR Î´ (TFA-d, 75 MHz) ppm: 54.9, 55.2 (MeOÃ—2), 111.2 (C-2â€²), 112.0 (C-5â€²), 116.3 (C-6), 119.2 (C-6â€²), 120.5 (C-1â€²), 121.1 (C-4-a), 124.7 (C-8), 126.6 (C-5), 136.4 (C-3), 138.7 (C-7), 148.0 (C-8a), 152.6 (C-4â€²), 153.9 (C-3â€²), 155.8 (C-2), 170.1 (C-4).

Example 2Synthesis of 2-(4-benzyloxy-3-methoxyphenyl)-6-bromo-3-hydroxy-4H-chromen-4-one (1b)

The compound 2-(4-benzyloxy-3-methoxyphenyl)-6-bromo-3-hydroxy-4H-chromen-4-one (1b) was prepared by the method of Example 1, but using 4-(benzyloxy)-3-methoxybenzaldehyde instead of 3,4-dimethoxy benzaldehyde.

Molecular Weight: 453.28 (C₂₃H₁₇BrO₅).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 3.95 (s, 3H, MeO), 5.25 (s, 2H, CH₂Ph), 6.97 (d, 1H, J=8.6, H-5â€²), 7.37 (m, 7H, H-7,8, CH₂Ph), 7.73 (d, 1H, J=2.1, H-2â€²), 7.79 (dd, 1H, J=8.6, 2.1, H-6â€²), 8.32 (d, 1H, J=2.0, H-5).

¹³C-NMR Î´ (CDCl₃, 75 MHz) ppm: 55.4 (MeO), 70.0 (CH₂Ph), 110.5 (C-2â€²), 112.5 (C-5â€²), 117.0 (C-6), 119.3 (C-6â€²), 120.7 (C-8), 121.3 (C-1â€²), 122.8 (C-4-a), 126.5 (CH₂Ph), 127.1, 127.3 (C-5, CH₂Ph), 127.9 (CH₂Ph), 135.6 (C-7), 135.7 (C-3), 137.1 (CH₂Ph), 144.8 (C-8a), 148.6 (C-4â€²), 149.4 (C-3â€²), 153.1 (C-2), 171.1 (C-4).

Example 3Synthesis of 2-(3-benzyloxy-4-methoxyphenyl)-6-bromo-3-hydroxy-4H-chromen-4-one (1c)

The compound 2-(3-benzyloxy-4-methoxyphenyl)-6-bromo-3-hydroxy-4H-chromen-4-one (1c) was prepared by the method of Example 1, but using 3-(benzyloxy)-4-methoxybenzaldehyde instead of 3,4-dimethoxy benzaldehyde.

Molecular Weight: 453.28 (C₂₃H₁₇BrO₅).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 3.97 (s, 3H, MeO), 5.25 (s, 2H, CH₂Ph), 7.02 (d, 1H, J=9.3, H-5â€²), 7.29-7.52 (m, 6H, H-8, CH₂Ph), 7.74 (dd, 1H, J=8.7, 2.4, H-7), 7.86 (m, 2H, H-2â€²,6â€²), 8.35 (d, 1H, J=2.4, H-5).

Example 4Synthesis of 7-(benzyloxy)-2-(4-(benzyloxy)-3-methoxyphenyl)-6-bromo-3-hydroxy-4H-chromen-4-one (1d)

The compound 7-(benzyloxy)-2-(4-(benzyloxy)-3-methoxyphenyl)-6-bromo-3-hydroxy-4H-chromen-4-one (1d) was prepared by the method of Example 1, but using 1-(4-(benzyloxy)-5-bromo-2-hydroxyphenyl)ethanone and 4-(benzyloxy)-3-methoxybenzaldehyde.

Molecular Weight: 559.40 (C₃₀H₂₃BrO₆).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 4.00 (s, 3H, MeO), 5.26 (m, 4H, 2*CH₂Ph), 7.02 (m, 2H, H-8.5â€²), 7.42 (m, 10H, 2*CH₂Ph), 7.74 (dd, 1H, J=8.6, 2.1, H-6â€²), 7.83 (m, 1H, H-2â€²), 8.43 (s, 1H, H-5).

Following the method of Example 1, the following compounds shown in Process A are prepared:

2-(4-(benzyloxy)-3-methoxyphenyl)-6-bromo-4-oxo-4H-chromen-3-yl acetate
2-(4-(benzyloxy)-3-methoxyphenyl)-6-bromo-4-oxo-4H-chromen-3-yl dimethylcarbamate
6-Bromo-2-(3,4-dimethoxyphenyl)-3-hydroxy-7-methoxy-4H-chromen-4-one
2-(4-Benzyloxy-3-methoxyphenyl)-6-bromo-3-hydroxy-7-methoxy-4H-chromen-4-one
6-Bromo-2-(3,4-dimethoxyphenyl)-3-hydroxy-8-methoxy-4H-chromen-4-one
2-(4-Benzyloxy-3-methoxyphenyl)-6-bromo-3-hydroxy-8-methoxy-4H-chromen-4-one
6-Bromo-2-(3,4-dimethoxyphenyl)-3-hydroxy-7,8-dimethoxy-4H-chromen-4-one
2-(4-Benzyloxy-3-methoxyphenyl)-6-bromo-3-hydroxy-7,8-dimethoxy-4H-chromen-4-one

Example 5Synthesis of 6-(12-(benzyloxy)dodec-1-ynyl)-2-(3,4-dimethoxyphenyl)-3-hydroxy-4H-chromen-4-one (2a)

To a solution of 6-bromo-2-(3,4-dimethoxyphenyl)-3-hydroxy-4H-chromen-4-one (0.50 g, 1.33 mmol, 1 eq) and tetrakis(triphenylphosphine)palladium (0.11 g, 0.09 mmol, 0.07 eq) in piperidine (2 mL) at 80Â° C., a solution of 1-((dodec-11-ynyloxy)methyl)benzene (0.54 g, 1.99 mmol, 1.5 eq) in piperidine (2 mL) was added. The reaction mixture was heated for 3 h at 80Â° C. and then quenched with saturated ammonium chloride (50 mL). The aqueous phase was extracted with dichloromethane (3*50 mL). The combined organic layers were washed with brine, dried over magnesium sulfate and concentrated in vacuo. The residue was purified by silica gel flash chromatography (heptanes/ethyl acetate: 7/3) to give 0.68 g (90%) of a yellow solid.

Molecular Weight: 568.70 (C₃₆H₄₀O₆).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 1.31 (s large, 12H, H-5â€³ to H-10â€³), 1.41 to 1.64 (m, 4H, H-4â€³,11â€³), 2.43 (t, 2H, J=6.9, H-3â€³), 3.46 (t, 2H, J=6.6, H-12â€³), 3.97 (s, 3H, MeO), 3.99 (s, 3H, MeO), 4.50 (s, 2H, CH₂Ph), 6.99 (s large, 1H, OH), 7.01 (d, 1H, J=8.6, H-5â€²), 7.33 (m, 5H, CH₂Ph), 7.49 (d, 1H, J=8.8, H-8), 7.66 (dd, 1H, J=8.8, 2.1, H-7), 7.83 (d, 1H, J=2.0, H-2â€²), 7.87 (dd, 1H, J=8.6, 2.0, H-6â€²), 8.24 (d, 1H, J=2.1, H-5).

¹³C-NMR Î´ (CDCl₃, 75 MHz) ppm: 19.4 (C-3â€³), 26.2 (C-10â€³), 28.6 to 29.8 (C-4â€³ to C-9â€³, C-11â€³), 56.0 (MeOÃ—2), 70.5 (C-12â€³), 72.8 (CH₂Ph), 79.1 (C-1â€³), 91.8 (C-2â€³), 110.7, 110.9 (C-2â€², C-5â€²), 118.2 (C-8), 120.5, 120.8 (C-6, C-1â€²), 121.5 (C-6â€²), 123.5 (C-4-a), 127.4 (C-5), 127.6, 128.3 (CH₂Ph), 136.3 (C-7), 137.8 (C-3), 138.7 (CH₂Ph), 145.1 (C-8a), 148.9 (C-4â€²), 150.8 (C-3â€²), 154.2 (C-2), 172.5 (C-4).

Example 6Synthesis of 2-(4-benzyloxy-3-methoxyphenyl)-6-(12-benzyloxydodec-1-ynyl)-3-hydroxy-4H-chromen-4-one (2b)

The compound 2-(4-benzyloxy-3-methoxyphenyl)-6-(12-benzyloxydodec-1-ynyl)-3-hydroxy-4H-chromen-4-one (2b) was prepared by the method of Example 5, but using 2-(4-benzyloxy-3-methoxyphenyl)-6-bromo-3-hydroxy-4H-chromen-4-one instead of 6-bromo-2-(3,4-dimethoxyphenyl)-3-hydroxy-4H-chromen-4-one.

Molecular Weight: 644.80 (C₄₂H₄₄O₆).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 1.26 (s large, 12H, H-5â€³ to H-10â€³), 1.36 to 1.64 (m, 4H, H-4â€³,11â€³), 2.43 (t, 2H, J=7.0, H-3â€³), 3.47 (t, 2H, J=6.7, H-12â€³), 4.00 (s, 3H, MeO), 4.50 (s, 2H, CH₂Ph), 5.26 (s, 2H, CH₂Ph), 6.96 (s large, 1H, OH), 7.02 (d, 1H, J=8.7, H-5â€²), 7.28 to 7.50 (m, 11H, H-8, CH₂Ph), 7.66 (dd, 1H, J=8.7, 2.0, H-7), 7.80 (dd, 1H, J=8.7, 2.0, H-6â€²), 7.85 (d, 1H, J=2.0, H-2â€²), 8.25 (d, 1H, J=2.0, H-5).

¹³C-NMR Î´ (CDCl₃, 75 MHz) ppm: 18.6 (C-3â€³), 25.4 (C-10â€³), 27.9 to 29.0 (C-4â€³ to C-9â€³, C-11â€³), 55.4 (MeO), 69.8 (C-12â€³), 70.0, 72.1 (CH₂PhÃ—2), 79.1 (C-1â€³), 91.1 (C-2â€³), 110.4 (C-2â€²), 112.5 (C-5â€²), 117.5 (C-8), 119.8, 120.1 (C-6, C-1â€²), 120.6 (C-6â€²), 123.2 (C-4-a), 126.4 (C-5), 126.7, 126.8, 127.3, 127.5 (CH₂Ph), 135.5 (C-7), 137.0 (C-3), 137.8 (CH₂Ph), 144.5 (C-8a), 148.6 (C-4â€²), 149.5 (C-3â€²), 153.8 (C-2), 172.1 (C-4).

Example 7Synthesis of 2-(4-benzyloxy-3-methoxyphenyl)-6-(13-benzyloxytridec-1-ynyl)-3-hydroxy-4H-chromen-4-one (2c)

The compound 2-(4-benzyloxy-3-methoxyphenyl)-6-(13-benzyloxytridec-1-ynyl)-3-hydroxy-4H-chromen-4-one (2c) was prepared by the method of Example 6, but using 1-((tridec-12-ynyloxy)methyl)benzene instead of 1-((dodec-11-ynyloxy)methyl)benzene.

Molecular Weight: 658.82 (C₄₃H₄₆O₆).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 1.25 (s large, 14H, H-5â€³ to H-11â€³), 1.31 to 1.63 (m, 4H, H-4â€³,12â€³), 2.47 (t, 2H, J=7.0, H-3â€³), 3.45 (t, 2H, J=6.7, H-13â€³), 3.99 (s, 3H, MeO), 4.52 (s, 2H, CH₂Ph), 5.25 (s, 2H, CH₂Ph), 6.92 (s large, 1H, OH), 7.02 (d, 1H, J=8.7, H-5â€²), 7.26 to 7.52 (m, 11H, H-8, CH₂Ph), 7.68 (dd, 1H, J=8.7, 2.0, H-7), 7.78 (dd, 1H, J=8.7, 2.0, H-6â€²), 7.83 (d, 1H, J=2.0, H-2â€²), 8.26 (d, 1H, J=2.0, H-5)

¹³C-NMR Î´ (CDCl₃, 75 MHz) ppm: 18.5 (C-3â€³), 25.3 (C-11â€³), 27.7 to 29.2 (C-4â€³ to C-10â€³, C-12â€³), 55.3 (MeO), 69.6 (C-13â€³), 70.1, 72.1 (CH₂PhÃ—2), 79.3 (C-1â€³), 91.2 (C-2â€³), 110.3 (C-2â€²), 112.4 (C-5â€²), 117.3 (C-8), 119.7, 120.2 (C-6, C-1â€²), 120.5 (C-6â€²), 123.1 (C-4-a), 126.5 (C-5), 126.7, 126.8, 127.2, 127.6 (CH₂Ph), 135.5 (C-7), 137.2 (C-3), 137.9 (CH₂Ph), 144.3 (C-8a), 148.5 (C-4â€²), 149.4 (C-3â€²), 153.7 (C-2), 172.3 (C-4).

Example 8Synthesis of 2-(3-benzyloxy-4-methoxyphenyl)-6-(12-benzyloxydodec-1-ynyl)-3-hydroxy-4H-chromen-4-one (2d)

The compound 2-(3-benzyloxy-4-methoxyphenyl)-6-(12-benzyloxydodec-1-ynyl)-3-hydroxy-4H-chromen-4-one (2d) was prepared by the method of Example 5, but using 2-(3-benzyloxy-4-methoxyphenyl)-6-bromo-3-hydroxy-4H-chromen-4-one instead of 6-bromo-2-(3,4-dimethoxyphenyl)-3-hydroxy-4H-chromen-4-one.

Molecular Weight: 644.80 (C₄₂H₄₄O₆).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 1.26 (s large, 12H, H-5â€³ to H-10â€³), 1.36 to 1.64 (m, 4H, H-4â€³,11â€³), 2.43 (t, 2H, J=6.9, H-3â€³), 3.47 (t, 2H, J=6.6, H-12â€³), 3.97 (s, 3H, MeO), 4.50 (s, 2H, CH₂Ph), 5.25 (s, 2H, CH₂Ph), 6.93 (s large, 1H, OH), 7.02 (d, 1H, J=9.2, H-5â€²), 7.29 to 7.45 (m, 10H, 2*CH₂Ph), 7.44 (d, 1H, J=8.7, H-8), 7.65 (dd, 1H, J=8.7, 2.0, H-7), 7.80 (m, 2H, H-2â€²,6â€²), 8.24 (d, 1H, J=2.0, H-5).

Example 9Synthesis of 7-(benzyloxy)-2-(4-benzyloxy-3-methoxyphenyl)-6-(12-benzyloxydodec-1-ynyl)-3-hydroxy-4H-chromen-4-one (2e)

The compound 2e was prepared by the method of Example 5, but using 7-(benzyloxy)-2-(4-(benzyloxy)-3-methoxyphenyl)-6-bromo-3-hydroxy-4H-chromen-4-one instead of 6-bromo-2-(3,4-dimethoxyphenyl)-3-hydroxy-4H-chromen-4-one.

Molecular Weight: 750.92 (C₄₉H₅₀O₇).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 1.28 (s large, 12H, H-5â€³ to H-10â€³), 1.35 to 1.61 (m, 4H, H-4â€³,11â€³), 2.47 (t, 2H, J=7.0, H-3â€³), 3.46 (t, 2H, J=6.6, H-12â€³), 3.99 (s, 3H, MeO), 4.50 (s, 2H, CH₂Ph), 5.24 (s, 4H, 2*CH₂Ph), 6.93 (s, 1H, H-8), 7.00 (d, 1H, J=8.6, H-5â€²), 7.30 to 7.52 (m, 15H, 3*CH₂Ph), 7.73 (dd, 1H, J=8.6, 1.8, H-6â€²), 7.82 (d, 1H, J=1.8, H-2â€²), 8.25 (s, 1H, H-5).

Example 10Synthesis of 2-(4-benzyloxy-3-methoxyphenyl)-6-(13-benzyloxy tridec-1-ynyl)-3-methoxy-4H-chromen-4-one (2f)

The compound 2f was prepared by the method of Example 5, but using 2-(4-benzyloxy-3-methoxyphenyl)-6-bromo-3-methoxy-4H-chromen-4-one instead of 6-bromo-2-(3,4-dimethoxyphenyl)-3-hydroxy-4H-chromen-4-one.

Molecular Weight: 672.85 (C₄₄H₄₈O₆).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 1.26 (s large, 14H, H-5â€³ to H-11â€³), 1.36 to 1.61 (m, 4H, H-4â€³,12â€³), 2.43 (t, 2H, J=7.2, H-3â€³), 3.47 (t, 2H, J=6.6, H-13â€³), 3.88 (s, 3H, MeO), 3.97 (s, 3H, MeO), 4.50 (s, 2H, CH₂Ph), 5.26 (s, 2H, CH₂Ph), 6.99 (d, 1H, J=8.6, H-5â€²), 7.28 to 7.45 (m, 11H, H-8, CH₂Ph), 7.64 (dd, 1H, J=8.7, 2.1, H-7), 7.68 (dd, 1H, J=8.6, 2.1, H-6â€²), 7.75 (d, 1H, J=2.1, H-2â€²), 8.28 (d, 1H, J=2.1, H-5).

Following the method of Example 5, the following compounds shown in Process A are prepared:

2-(4-Benzyloxy-3-methoxyphenyl)-6-(12-benzyloxydodec-1-ynyl)-4-oxo-4H-chromen-3-yl acetate
2-(4-Benzyloxy-3-methoxyphenyl)-6-(13-benzyloxytridec-1-ynyl)-4-oxo-4H-chromen-3-yl acetate
2-(4-Benzyloxy-3-methoxyphenyl)-6-(12-benzyloxydodec-1-ynyl)-4-oxo-4H-chromen-3-yl dimethylcarbamate
2-(4-Benzyloxy-3-methoxyphenyl)-6-(13-benzyloxytridec-1-ynyl)-4-oxo-4H-chromen-3-yl dimethylcarbamate
6-(12-Benzyloxydodec-1-ynyl)-2-(3,4-dimethoxyphenyl)-3-hydroxy-7-methoxy-4H-chromen-4-one
6-(13-Benzyloxytridec-1-ynyl)-2-(3,4-dimethoxyphenyl)-3-hydroxy-7-methoxy-4H-chromen-4-one
2-(4-Benzyloxy-3-methoxyphenyl)-6-(12-benzyloxydodec-1-ynyl)-3-hydroxy-7-methoxy-4H-chromen-4-one
2-(4-Benzyloxy-3-methoxyphenyl)-6-(13-benzyloxytridec-1-ynyl)-3-hydroxy-7-methoxy-4H-chromen-4-one
6-(12-benzyloxydodec-1-ynyl)-2-(3,4-dimethoxyphenyl)-3-hydroxy-8-methoxy-4H-chromen-4-one
6-(13-benzyloxytridec-1-ynyl)-2-(3,4-dimethoxyphenyl)-3-hydroxy-8-methoxy-4H-chromen-4-one
2-(4-Benzyloxy-3-methoxyphenyl)-6-(12-benzyloxydodec-1-ynyl)-3-hydroxy-8-methoxy-4H-chromen-4-one
2-(4-Benzyloxy-3-methoxyphenyl)-6-(13-benzyloxytridec-1-ynyl)-3-hydroxy-8-methoxy-4H-chromen-4-one
6-(12-Benzyloxydodec-1-ynyl)-2-(3,4-dimethoxyphenyl)-3-hydroxy-7,8-dimethoxy-4H-chromen-4-one
6-(13-Benzyloxytridec-1-ynyl)-2-(3,4-dimethoxyphenyl)-3-hydroxy-7,8-dimethoxy-4H-chromen-4-one
2-(4-Benzyloxy-3-methoxyphenyl)-6-(12-benzyloxydodec-1-ynyl)-3-hydroxy-7,8-dimethoxy-4H-chromen-4-one
2-(4-Benzyloxy-3-methoxyphenyl)-6-(13-benzyloxytridec-1-ynyl)-3-hydroxy-7,8-dimethoxy-4H-chromen-4-one

Example 11Synthesis of 2-(3,4-dimethoxyphenyl)-3-hydroxy-6-(12-hydroxydodecyl)-4H-chromen-4-one (3a)

To a solution of 6-(12-(benzyloxy)dodec-1-ynyl)-2-(3,4-dimethoxyphenyl)-3-hydroxy-4H-chromen-4-one (0.66 g, 1.16 mmol) in THF/acetic acid (9/1) (4 mL), was added 10% Pd/C (0.13 g, 20% w/w). The reaction mixture was then stirred under hydrogen atmosphere for 5 h at room temperature. The solution was filtered through Celite and concentrated in vacuo. The residue was purified by silica gel flash chromatography (heptanes/ethyl acetate: 5/5) to give 0.49 g (87%) of a yellow solid.

Molecular Weight: 482.61 (C₂₉H₃₈O₆).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 1.26 (s large, 16H, H-3â€³ to H-10â€³), 1.54 to 1.67 (m, 4H, H-2â€³,11â€³), 2.73 (t, 2H, J=7.8, H-1â€³), 3.64 (t, 2H, J=6.5, H-12â€³), 3.97 (s, 3H, MeO), 3.99 (s, 3H, MeO), 7.02 (d, 1H, J=8.6, H-5â€²), 7.51 (s, 2H, H-7, H-8), 7.85 (d, 1H, J=2.0, H-2â€²), 7.89 (dd, 1H, J=8.6, 2.0, H-6â€²), 8.02 (s, 1H, H-5).

¹³C-NMR Î´ (CDCl₃, 75 MHz) ppm: 25.7 (C-10â€³), 29.1 (C-3â€³), 29.4 to 29.7 (C-4â€³ to C-9â€³), 31.3 (C-2â€³), 32.8 (C-11â€³), 35.3 (C-1â€³), 56.0 (MeOÃ—2), 63.1 (C-12â€³), 110.7, 110.9 (C-2â€², C-5â€²), 117.9 (C-8), 120.4 (C-1â€²), 121.4 (C-6â€²), 123.4 (C-4-a), 123.9 (C-5), 134.3 (C-7), 137.7 (C-6), 139.5 (C-3), 145.0 (C-8a), 148.8 (C-4â€²), 150.7 (C-3â€²), 153.8 (C-2), 173.2 (C-4).

Example 12Synthesis of 3-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-(12-hydroxydodecyl)-4H-chromen-4-one (3b)

The compound 3b was prepared by the method of Example 10, but using 2-(4-benzyloxy-3-methoxyphenyl)-6-(12-benzyloxydodec-1-ynyl)-3-hydroxy-4H-chromen-4-one instead of 6-(12-(benzyloxy)dodec-1-ynyl)-2-(3,4-dimethoxy phenyl)-3-hydroxy-4H-chromen-4-one.

Molecular Weight: 468.58 (C₂₈H₃₆O₆).

¹H-NMR Î´ (CD₃OD, 300 MHz) ppm (J in Hz): 1.26 (s large, 16H, H-3â€³ to H-10â€³), 1.52 to 1.70 (m, 4H, H-2â€³,11â€³), 2.73 (t, 2H, J=7.4, H-1â€³), 3.53 (t, 2H, J=6.6, H-12â€³), 4.00 (s, 3H, MeO), 7.07 (d, 1H, J=8.6, H-5â€²), 7.51 (m, 2H, H-7, H-8), 7.82 (dd, 1H, J=8.6, 2.0, H-6â€²), 7.87 (d, 1H, J=2.0, H-2â€²), 8.02 (s, 1H, H-5).

¹³C-NMR Î´ (CD₃OD, 75 MHz) ppm: 25.7 (C-10â€³), 29.1 (C-3â€³), 29.4 to 29.6 (C-4â€³ to C-9â€³), 31.3 (C-2â€³), 32.8 (C-11â€³), 35.3 (C-1â€³), 56.1 (MeO), 63.1 (C-12â€³), 110.4 (C-2â€²), 114.6 (C-5â€²), 117.9 (C-8), 120.4 (C-1â€²), 121.8 (C-6â€²), 123.4 (C-4-a), 123.9 (C-5), 134.3 (C-7), 137.6 (C-6), 139.5 (C-3), 145.2 (C-8a), 146.4 (C-4â€²), 147.6 (C-3â€²), 153.7 (C-2), 173.3 (C-4).

Example 13Synthesis of 3-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-(13-hydroxytridecyl)-4H-chromen-4-one (3c)

The compound 3c was prepared by the method of Example 12, but using 2-(4-benzyloxy-3-methoxyphenyl)-6-(13-benzyloxytridec-1-ynyl)-3-hydroxy-4H-chromen-4-one instead of 2-(4-benzyloxy-3-methoxyphenyl)-6-(12-benzyloxydodec-1-ynyl)-3-hydroxy-4H-chromen-4-one.

Molecular Weight: 482.61 (C₂₉H₃₈O₆).

¹H-NMR Î´ (CD₃OD, 300 MHz) ppm (J in Hz): 1.22 (s large, 16H, H-3â€³ to H-11â€³), 1.49 to 1.72 (m, 4H, H-2â€³,12â€³), 2.71 (t, 2H, J=7.8, H-1â€³), 3.52 (t, 2H, J=6.9, H-13â€³), 3.94 (s, 3H, MeO), 6.96 (d, 1H, J=8.6, H-5â€²), 7.50 (m, 2H, H-7, H-8), 7.77 (dd, 1H, J=8.6, 2.0, H-6â€²), 7.84 (d, 1H, J=2.0, H-2â€²), 7.93 (s, 1H, H-5).

¹³C-NMR Î´ (CD₃OD, 75 MHz) ppm: 25.6 (C-11â€³), 29.3 (C-3â€³), 29.3 to 29.8 (C-4â€³ to C-10â€³), 31.2 (C-2â€³), 32.7 (C-12â€³), 35.6 (C-1â€³), 56.3 (MeO), 63.2 (C-13â€³), 110.9 (C-2â€²), 114.3 (C-5â€²), 118.0 (C-8), 120.3 (C-1â€²), 121.6 (C-6â€²), 123.2 (C-4-a), 123.7 (C-5), 134.1 (C-7), 137.4 (C-6), 139.2 (C-3), 145.4 (C-8a), 146.6 (C-4â€²), 147.9 (C-3â€²), 153.5 (C-2), 173.1 (C-4).

Example 14Synthesis of 3-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-6-(12-hydroxydodecyl)-4H-chromen-4-one (3d)

The compound 3d was prepared by the method of Example 10, but using 2-(3-benzyloxy-4-methoxyphenyl)-6-(12-benzyloxydodec-1-ynyl)-3-hydroxy-4H-chromen-4-one instead of 6-(12-(benzyloxy)dodec-1-ynyl)-2-(3,4-dimethoxy phenyl)-3-hydroxy-4H-chromen-4-one.

Molecular Weight: 468.58 (C₂₈H₃₆O₆).

¹H-NMR Î´ (CD₃OD, 300 MHz) ppm (J in Hz): 1.22 (s large, 16H, H-3â€³ to H-10â€³), 1.49 to 1.63 (m, 4H, H-2â€³,11â€³), 2.69 (t, 2H, J=7.8, H-1â€³), 3.52 (t, 2H, J=6.9, H-12â€³), 3.92 (s, 3H, MeO), 6.97 (d, 1H, J=9.0, H-5â€²), 7.48 (m, 2H, H-7, H-8), 7.80 (m, 2H, H-2â€²,6â€²), 7.91 (d, 1H, J=2.0, H-5).

¹³C-NMR Î´ (CD₃OD, 75 MHz) ppm: 26.2 (C-10â€³), 29.5 (C-3â€³), 29.8 to 29.9 (C-4â€³ to C-9â€³), 31.7 (C-2â€³), 32.9 (C-11â€³), 35.7 (C-1â€³), 56.1 (MeO), 62.7 (C-12â€³), 111.4 (C-5â€²), 115.0 (C-2â€²), 118.4 (C-8), 121.1 (C-4-a), 121.2 (C-6â€²), 123.9 (C-5), 124.5 (C-1â€²), 134.7 (C-7), 138.4 (C-6), 139.9 (C-3), 146.3 (C-8a), 146.6 (C-3â€²), 149.7 (C-4â€²), 154.1 (C-2), 173.9 (C-4).

Example 15Synthesis of 3,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-(12-hydroxydodecyl)-4H-chromen-4-one (3e)

The compound 3e was prepared by the method of Example 10, but using 7-(benzyloxy)-2-(4-benzyloxy-3-methoxyphenyl)-6-(12-benzyloxydodec-1-ynyl)-3-hydroxy-4H-chromen-4-one instead of 6-(12-(benzyloxy)dodec-1-ynyl)-2-(3,4-dimethoxy phenyl)-3-hydroxy-4H-chromen-4-one.

Molecular Weight: 484.58 (C₂₈H₃₆O₇).

¹H-NMR Î´ (CD₃OD, 300 MHz) ppm (J in Hz): 1.23 (s large, 16H, H-3â€³ to H-10â€³), 1.49 to 1.60 (m, 4H, H-2â€³,11â€³), 2.65 (t, 2H, J=7.7, H-1â€³), 3.52 (t, 2H, J=6.8, H-12â€³), 3.93 (s, 3H, MeO), 6.86 (s, 1H, H-8), 6.94 (d, 1H, J=8.5, H-5â€²), 7.69 (dd, 1H, J=8.5, 2.0, H-6â€²), 7.81 (d, 1H, J=2.0, H-2â€²), 7.82 (s, 1H, H-5).

Example 16Synthesis of 2-(4-hydroxy-3-methoxyphenyl)-6-(13-hydroxy tridecyl)-3-methoxy-4H-chromen-4-one (3f)

The compound 3f was prepared by the method of Example 10, but using 2-(4-benzyloxy-3-methoxyphenyl)-6-(13-benzyloxy tridec-1-ynyl)-3-methoxy-4H-chromen-4-one instead of 6-(12-(benzyloxy)dodec-1-ynyl)-2-(3,4-dimethoxy phenyl)-3-hydroxy-4H-chromen-4-one.

Molecular Weight: 496.63 (C₃₀H₄₀O₆).

¹H-NMR Î´ (CD₃OD, 300 MHz) ppm (J in Hz): 1.24 (s large, 16H, H-3â€³ to H-11â€³), 1.50 to 1.65 (m, 4H, H-2â€³,12â€³), 2.72 (t, 2H, J=7.8, H-1â€³), 3.52 (t, 2H, J=6.9, H-13â€³), 3.79 (s, 3H, MeO), 3.94 (s, 3H, MeO), 6.96 (d, 1H, J=8.4, H-5â€²), 7.51 (m, 2H, H-7, H-8), 7.68 (dd, 1H, J=8.4, 2.1, H-6â€²), 7.72 (d, 1H, J=2.1, H-2â€²), 7.96 (d, 1H, J=1.8, H-5).

¹³C-NMR Î´ (CD₃OD, 75 MHz) ppm: 26.3 (C-11â€³), 29.7 (C-3â€³), 30.0 to 30.2 (C-4â€³ to C-10â€³), 31.9 (C-2â€³), 33.0 (C-12â€³), 35.9 (C-1â€³), 56.4 (MeO), 60.3 (MeO), 62.8 (C-13â€³), 112.4 (C-2â€²), 115.9 (C-5â€²), 118.5 (C-8), 122.6 (C-4-a), 123.4 (C-6â€²), 124.1 (C-1â€²), 124.5 (C-5), 135.1 (C-7), 140.6 (C-3), 141.1 (C-6), 148.1 (C-8a), 150.1 (C-4â€²), 154.4 (C-3â€²), 157.5 (C-2), 176.4 (C-4).

Following the method of Example 11, the following compounds shown in Process A are prepared:

2-(4-Hydroxy-3-methoxyphenyl)-6-(12-hydroxydodecyl)-4-oxo-4H-chromen-3-yl acetate
2-(4-Hydroxy-3-methoxyphenyl)-6-(13-hydroxytridecyl)-4-oxo-4H-chromen-3-yl acetate
2-(4-Hydroxy-3-methoxyphenyl)-6-(12-hydroxydodecyl)-4-oxo-4H-chromen-3-yl dimethylcarbamate
2-(4-Hydroxy-3-methoxyphenyl)-6-(13-hydroxytridecyl)-4-oxo-4H-chromen-3-yl dimethylcarbamate
2-(3,4-Dimethoxyphenyl)-3-hydroxy-6-(12-hydroxydodecyl)-7-methoxy-4H-chromen-4-one
2-(3,4-Dimethoxyphenyl)-3-hydroxy-6-(13-hydroxytridecyl)-7-methoxy-4H-chromen-4-one
3-Hydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-(12-hydroxydodecyl)-7-methoxy-4H-chromen-4-one
3-Hydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-(13-hydroxytridecyl)-7-methoxy-4H-chromen-4-one
2-(3,4-Dimethoxyphenyl)-3-hydroxy-6-(12-hydroxydodecyl)-8-methoxy-4H-chromen-4-one
2-(3,4-Dimethoxyphenyl)-3-hydroxy-6-(13-hydroxytridecyl)-8-methoxy-4H-chromen-4-one
3-Hydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-(12-hydroxydodecyl)-8-methoxy-4H-chromen-4-one
3-Hydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-(13-hydroxytridecyl)-8-methoxy-4H-chromen-4-one
2-(3,4-Dimethoxyphenyl)-3-hydroxy-6-(12-hydroxydodecyl)-7,8-dimethoxy-4H-chromen-4-one
2-(3,4-Dimethoxyphenyl)-3-hydroxy-6-(13-hydroxytridecyl)-7,8-dimethoxy-4H-chromen-4-one
3-Hydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-(12-hydroxydodecyl)-7,8-dimethoxy-4H-chromen-4-one
3-Hydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-(13-hydroxytridecyl)-7,8-dimethoxy-4H-chromen-4-one

Example 17Synthesis of 2-(3,4-dihydroxyphenyl)-3-hydroxy-6-(12-hydroxydodecyl)-4H-chromen-4-one (4)

To a solution of 2-(3,4-dimethoxyphenyl)-3-hydroxy-6-(12-hydroxydodecyl)-4H-chromen-4-one (0.20 g, 0.41 mmol, 1 eq) in dichloromethane (10 mL) at 0Â° C., was added boron tribromide in dichloromethane (1.0 M, 2.5 mL, 2.49 mmol, 6 eq). The mixture was warmed to room temperature and then stirred for 22 h. The reaction mixture was then cooled to 0Â° C. and methanol (10 mL) was added. The reaction mixture was heated to reflux for 2 h, and then concentrated in vacuo to give a yellow solid. Water (50 mL) was added and the aqueous phase was extracted with ethyl acetate (3*50 mL). The combined organic layers were washed with brine, dried over magnesium sulfate and concentrated in vacuo. The residue was recrystallized from ethanol to give 0.10 g (55%) of a yellow solid.

Molecular Weight: 454.56 (C₂₇H₃₄O₆).

¹H-NMR Î´ (DMSO-d6, 300 MHz) ppm (J in Hz): 1.22 (s large, 16H, H-3â€³ to H-10â€³), 1.58 to 1.80 (m, 4H, H-2â€³,11â€³), 2.70 (t, 2H, J=7.5, H-1â€³), 3.37 (t, 2H, J=6.8, H-12â€³), 6.89 (d, 1H, J=8.6, H-5â€²), 7.59 (dd, 1H, J=8.6, 2.1, H-6â€²), 7.61 (s, 2H, H-7, H-8), 7.74 (d, 1H, J=2.1, H-2â€²), 7.86 (s, 1H, H-5), 9.23, 9.28, 9.55 (3s, OHÃ—3).

¹³C-NMR Î´ (DMSO-d₆, 75 MHz) ppm: 28.0 (C-10â€³), 28.6 (C-3â€³), 29.0 to 29.4 (C-4â€³ to C-9â€³), 31.3 (C-2â€³), 32.7 (C-11â€³), 35.6 (C-1â€³), 115.7, 116.0 (C-2â€², C-5â€²), 118.5 (C-8), 120.4 (C-6â€²), 121.5 (C-1â€²), 122.8 (C-4-a), 123.6 (C-5), 134.4 (C-7), 138.3 (C-6), 139.1 (C-3), 145.5 (C-4â€²), 146.4 (C-8a), 148.0 (C-3â€²), 153.3 (C-2), 172.8 (C-4).

Following the method of Example 17, the following compounds shown in Process A are prepared:

2-(3,4-Dihydroxyphenyl)-3,7-dihydroxy-6-(12-hydroxydodecyl)-4H-chromen-4-one
2-(3,4-Dihydroxyphenyl)-3,7-dihydroxy-6-(13-hydroxytridecyl)-4H-chromen-4-one
2-(3,4-Dihydroxyphenyl)-3,8-dihydroxy-6-(12-hydroxydodecyl)-4H-chromen-4-one
2-(3,4-Dihydroxyphenyl)-3,8-dihydroxy-6-(13-hydroxytridecyl)-4H-chromen-4-one
2-(3,4-Dihydroxyphenyl)-3,7,8-trihydroxy-6-(12-hydroxydodecyl)-4H-chromen-4-one
2-(3,4-Dihydroxyphenyl)-3,7,8-trihydroxy-6-(13-hydroxytridecyl)-4H-chromen-4-one

Example Synthesis of Compound 10 of General Formula (I)Example 18Synthesis of 2-(3,4-dimethoxyphenyl)-5-hydroxy-3,7-dimethoxy-4H-chromen-4-one (5a)

To a solution of commercial quercetin dihydrate (5.0 g, 14.78 mmol, 1 eq) in acetone (200 mL), potassium carbonate (8.4 g, 60.60 mmol, 4.1 eq) and dimethylsulfate (5.61 mL, 59.12 mmol, 4 eq) were added under argon at 0Â° C. After vigorous stirring at reflux for 3 h, the resulting mixture was filtered and the filtrate concentrated in vacuo. The residue was recristallized from water/ethanol (1/1) (150 mL) then washed with diethyl ether (50 mL) to afford 3.23 g (61%) of a pale yellow solid.

Molecular Weight: 358.34 (C₁₉H₁₈O₇).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 3.86 (s, 3H, MeO), 3.87 (s, 3H, MeO), 3.96 (s, 3H, MeO), 3.97 (s, 3H, MeO), 6.35 (d, 1H, J=2.1, H-6), 6.44 (d, 1H, J=2.1, H-8), 6.98 (d, 1H, J=8.7, H-5â€²), 7.68 (d, 1H, J=2.1, H-2â€²), 7.73 (dd, 1H, J=8.7, 2.1, H-6â€²).

Example 19Synthesis of 2-(3,4-bisbenzyloxyphenyl)-3,7-bisbenzyloxy-5-hydroxy-4H-chromen-4-one (5b) and 3,7-bisbenzyloxy-2-(4-benzyloxy-3-hydroxyphenyl)-5-hydroxy-4H-chromen-4-one (5c)

To a solution of commercial quercetin dihydrate (10.0 g, 29.56 mmol, 1 eq) in DMF (250 mL), potassium carbonate (24.51 g, 0.15 mol, 5 eq) and benzyl bromide (12.6 mL, 0.11 mol, 3.5 eq) were added under argon at 0Â° C. After vigorous stirring at room temperature for 48 h, the resulting mixture quenched with distillated water (300 mL) then DMF was removed by distillation under reduced pressure. 150 mL of diethyl ether was added to the aqueous phase and the white precipitate obtained was filtered off and washed once with diethyl ether (75 mL) to give 10.31 g (53%) of tetrabenzyl derivative 5b.

Molecular Weight: 662.73 (C₄₃H₃₄O₇).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 5.00 (s, 2H, CH₂Ph), 5.06 (s, 2H, CH₂Ph), 5.13 (s, 2H, CH₂Ph), 5.25 (s, 2H, CH₂Ph), 6.44 (d, 1H, J=2.1, H-6), 6.46 (d, 1H, J=2.1, H-8), 6.97 (d, 1H, J=8.7, H-5â€²), 7.39 (m, 20H, 4*CH₂Ph), 7.56 (dd, 1H, J=8.7, 2.1, H-6â€²), 7.72 (d, 1H, J=2.1, H-2â€²), 12.72 (s, 1H, OH).

The filtrate was separated and the aqueous phase was extracted with ethyl acetate (3Ã—200 mL). The combined organic layers were washed with brine (150 mL), dried over magnesium sulfate and concentrated in vacuo to give brown oil. The residue was washed with ethanol-water (1/1) for 60 h to afford 3.54 g (21%) of a pale yellow solid 5c.

Molecular Weight: 572.60 (C₃₆H₂₈O₇).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 5.04 (s, 2H, CH₂Ph), 5.09 (s, 2H, CH₂Ph), 5.15 (s, 2H, CH₂Ph), 6.41 (d, 1H, J=2.1, H-6), 6.46 (d, 1H, J=2.1, H-8), 6.92 (d, 1H, J=8.7, H-5â€²), 7.39 (m, 16H, H-6â€², 3*CH₂Ph), 7.60 (d, 1H, J=2.1, H-2â€².

Example 20Synthesis of 2-(3,4-dimethoxyphenyl)-5-hydroxy-6-iodo-3,7-dimethoxy-4H-chromen-4-one (6a)

To a solution of 2-(3,4-dimethoxyphenyl)-5-hydroxy-3,7-dimethoxy-4H-chromen-4-one (6.05 g, 16.88 mmol, 1 eq) in dichloromethane-MeOH (5/2) (210 mL), calcium carbonate (11.8 g, 118.18 mmol, 7 eq) and benzyltrimethylammonium chloroiodate (BTMA-ICl₂) (5.88 g, 16.88 mmol, 1 eq) were added under argon at 0Â° C. After vigorous stirring at room temperature overnight, the resulting mixture was quenched with distillated water (150 mL), and the aqueous phase extracted with dichloromethane (3*150 mL). The combined organic layers were washed with brine, dried over magnesium sulfate and concentrated in vacuo. The residue was purified by silica gel column chromatography (heptanes/ethyl acetate: 7/3, 1/1 then 0/1) to give 4.93 g (60%) of a yellow solid.

Molecular Weight: 484.24 (C₁₉H₁₇IO₇).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 3.86 (s, 3H, MeO), 3.97 (s, 6H, MeOÃ—2), 3.99 (s, 3H, MeO), 6.49 (s, 1H, H-8), 6.98 (d, 1H, J=8.4, H-5â€²), 7.68 (d, 1H, J=2.1, H-2â€²), 7.73 (dd, 1H, J=8.4, 2.1, H-6â€²), 13.63 (s, 1H, OH).

¹³C-NMR Î´ (CDCl₃, 75 MHz) ppm: 56.2 (MeO), 56.3 (MeO), 57.1 (MeO), 60.4 (MeO), 69.6 (C-6), 90.4 (C-8), 106.2 (C-4-a), 111.0, 111.4 (C-2â€², C-5â€²), 122.4 (C-6â€²), 122.7 (C-1â€²), 139.1 (C-3), 148.9 (C-4â€²), 151.7 (C-3â€²), 156.3 (C-2), 157.2 (C-8a), 161.0 (C-5), 163.3 (C-7), 178.0 (C-4).

Example 21Synthesis of 2-(3,4-bisbenzyloxyphenyl)-3,7-bisbenzyloxy-5-hydroxy-6-iodo-4H-chromen-4-one (6b)

The compound 6b was prepared by the method of Example 18, but using 2-(3,4-bisbenzyloxyphenyl)-3,7-bisbenzyloxy-5-hydroxy-4H-chromen-4-one instead of 2-(3,4-dimethoxyphenyl)-5-hydroxy-3,7-dimethoxy-4H-chromen-4-one.

Molecular Weight: 788.62 (C₄₃H₃₃IO₇).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 4.99 (s, 2H, CH₂Ph), 5.05 (s, 2H, CH₂Ph), 5.24 (s, 2H, CH₂Ph), 5.25 (s, 2H, CH₂Ph), 6.47 (s, 1H, H-8), 6.96 (d, 1H, J=8.7, H-5â€²), 7.21-7.49 (m, 21H, H-6â€², 4*CH₂Ph), 7.70 (d, 1H, J=2.1, H-2â€²), 13.72 (s, 1H, OH).

¹³C-NMR Î´ (CDCl₃, 75 MHz) ppm: 70.2 (C-6), 71.0 (CH₂Ph), 71.3 (2*CH₂Ph), 74.5 (CH₂Ph), 91.6 (C-8), 106.3 (C-4-a), 113.8, 115.5 (C-2â€², C-5â€²), 122.8 (C-6â€²), 123.3 (C-1â€²), 127.0, 127.3, 127.5, 128.1, 128.2, 128.4, 128.5, 128.7, 128.8, 128.9, 130.0, 135.5, 136.3, 136.7, 137.0 (CH₂Ph) 137.5 (C-3), 148.4 (C-4â€²), 151.4 (C-3â€²), 156.7 (C-8a), 157.1 (C-5), 161.2 (C-2), 162.3 (C-7), 178.1 (C-4).

Example 22Synthesis of 3,7-bisbenzyloxy-2-(4-benzyloxy-3-hydroxyphenyl)-5-hydroxy-6-iodo-4H-chromen-4-one (6c)

The compound 6c was prepared by the method of Example 20, but using 3,7-bisbenzyloxy-2-(4-benzyloxy-3-hydroxyphenyl)-5-hydroxy-4H-chromen-4-one instead of 2-(3,4-dimethoxyphenyl)-5-hydroxy-3,7-dimethoxy-4H-chromen-4-one.

Molecular Weight: 698.5 (C₃₆H₂₇IO₇).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 5.06 (s, 2H, CH₂Ph), 5.16 (s, 2H, CH₂Ph), 5.19 (s, 2H, CH₂Ph), 6.47 (s, 1H, H-8), 6.90 (d, 1H, J=8.4, H-5â€²), 7.21-7.49 (m, 16H, H-6â€², 3*CH₂Ph), 7.62 (d, 1H, J=2.1, H-2â€²).

Example 23Synthesis of 2-(3,4-dimethoxyphenyl)-6-iodo-3,5,7-trimethoxy-4H-chromen-4-one (7a)

To a solution of 2-(3,4-dimethoxyphenyl)-5-hydroxy-6-iodo-3,7-dimethoxy-4H-chromen-4-one (1.0 g, 2.07 mmol, 1 eq) in THF (50 mL), potassium carbonate (0.56 g, 4.13 mmol, 2 eq) and dimethylsulfate (0.20 mL, 2.07 mmol, 1 eq) were added under argon at 0Â° C. After vigorous stirring at reflux overnight, the resulting mixture was quenched with distillated water (100 mL), and the aqueous phase was extracted with dichloromethane (3*100 mL). The combined organic layers were washed with brine, dried over magnesium sulfate and concentrated in vacuo. The residue was purified by silica gel column chromatography (heptanes/ethyl acetate: 7/3, 1/1 then 0/1) to give 0.68 g (66%) of a white solid.

Molecular Weight: 498.27 (C₂₀H₁₉IO₇).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 3.86 (s, 3H, MeO), 3.97 (s, 9H, MeOÃ—3), 4.00 (s, 3H, MeO), 6.73 (s, 1H, H-8), 6.98 (d, 1H, J=8.7, H-5â€²), 7.70 (d, 1H, J=2.1, H-2â€²), 7.71 (dd, 1H, J=8.7, 2.1, H-6â€²).

¹³C-NMR Î´ (CDCl₃, 75 MHz) ppm: 56.1 (MeO), 56.2 (MeO), 57.1 (MeO), 60.1 (MeO), 62.0 (MeO), 82.8 (C-6), 95.9 (C-8), 109.1 (C-4-a), 111.0, 111.4 (C-2â€², C-5â€²), 121.9 (C-6â€²), 123.1 (C-1â€²), 141.1 (C-3), 148.5 (C-4â€²), 151.2 (C-3â€²), 153.6 (C-2), 158.8 (C-8a), 160.4 (C-5), 161.9 (C-7), 172.6 (C-4).

Example 24Synthesis of 2-(3,4-bisbenzyloxyphenyl)-3,7-bisbenzyloxy-6-iodo-5-methoxy-4H-chromen-4-one (7b)

The compound 7b was prepared by the method of Example 23, but using 2-(3,4-bisbenzyloxyphenyl)-3,7-bisbenzyloxy-5-hydroxy-6-iodo-4H-chromen-4-one instead of 2-(3,4-dimethoxyphenyl)-5-hydroxy-6-iodo-3,7-dimethoxy-4H-chromen-4-one.

Molecular Weight: 802.65 (C₄₄H₃₅IO₇).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 4.03 (s, 3H, MeO), 5.01 (s, 2H, CH₂Ph), 5.09 (s, 2H, CH₂Ph), 5.28 (s, 4H, 2*CH₂Ph), 6.74 (s, 1H, H-8), 7.00 (d, 1H, J=8.7, H-5â€²), 7.25-7.53 (m, 21H, H-6â€², 4*CH₂Ph), 7.75 (d, 1H, J=1.8, H-2â€²).

¹³C-NMR Î´ (CDCl₃, 75 MHz) ppm: 62.1 (MeO), 71.0 (CH₂Ph), 71.1 (CH₂Ph), 71.5 (CH₂Ph), 74.3 (CH₂Ph), 83.4 (C-6), 97.1 (C-8), 113.7 (C-4-a), 113.8, 115.4 (C-2â€², C-5â€²), 122.3 (C-6â€²), 123.6 (C-1â€²), 127.0, 127.2, 127.4, 127.9, 128.0, 128.2, 128.3, 128.5, 128.6, 128.8, 129.0, 135.4, 136.8, 136.9 137.1 (CH₂Ph) 139.5 (C-3), 148.3 (C-4â€²), 150.9 (C-3â€²), 154.0 (C-8a), 158.6 (C-5), 160.5 (C-2), 160.8 (C-7), 172.7 (C-4).

Example 25Synthesis of 3,7-bisbenzyloxy-2-(4-benzyloxy-3-methoxyphenyl)-6-iodo-5-methoxy-4H-chromen-4-one (7c)

The compound 7c was prepared by the method of Example 23, but using 3,7-bisbenzyloxy-2-(4-benzyloxy-3-hydroxyphenyl)-5-hydroxy-6-iodo-4H-chromen-4-one instead of 2-(3,4-dimethoxyphenyl)-5-hydroxy-6-iodo-3,7-dimethoxy-4H-chromen-4-one.

Molecular Weight: 726.55 (C₃₈H₃₁IO₇).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 3.70 (s, 3H, MeO), 4.00 (s, 3H, MeO), 5.06 (s, 2H, CH₂Ph), 5.24 (s, 4H, 2*CH₂Ph), 6.76 (s, 1H, H-8), 6.94 (d, 1H, J=8.7, H-5â€²), 7.32-7.50 (m, 16H, H-6â€², 3*CH₂Ph), 7.65 (d, 1H, J=2.1, H-2â€²).

¹³C-NMR Î´ (CDCl₃, 75 MHz) ppm: 56.0 (MeO), 62.1 (MeO), 70.9 (CH₂Ph), 71.5 (CH₂Ph), 74.5 (CH₂Ph), 83.4 (C-6), 97.2 (C-8), 112.4, 113.0 (C-2â€², C-5â€²), 113.7 (C-4-a), 121.7 (C-6â€²), 123.5 (C-1â€²), 127.1, 127.4, 127.5, 128.2, 128.3, 128.4, 128.8, 128.9, 135.4, 136.6, 136.8 (CH₂Ph) 139.7 (C-3), 149.1 (C-4â€²), 150.2 (C-3â€²), 154.2 (C-8a), 158.7 (C-5), 160.5 (C-2), 160.9 (C-7), 172.7 (C-4).

Example 26Synthesis of 2-(3,4-bisbenzyloxyphenyl)-3,5,7-trisbenzyloxy-6-iodo-4H-chromen-4-one (7d)

The compound 7d was prepared by the method of Example 23, but using 2-(3,4-bisbenzyloxyphenyl)-3,7-bisbenzyloxy-5-hydroxy-6-iodo-4H-chromen-4-one instead of 2-(3,4-dimethoxyphenyl)-5-hydroxy-6-iodo-3,7-dimethoxy-4H-chromen-4-one and benzyl bromide instead of dimethylsulfate.

Molecular Weight: 878.77 (C₅₀H₃₉IO₇).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 4.99 (s, 2H, CH₂Ph), 5.08 (s, 2H, CH₂Ph), 5.12 (s, 2H, CH₂Ph), 5.25 (s, 4H, 2*CH₂Ph), 6.74 (s, 1H, H-8), 6.97 (d, 1H, J=8.7, H-5â€²), 7.21-7.56 (m, 25H, 5*CH₂Ph), 7.73 (d, 1H, J=2.1, H-2â€²), 7.87 (dd, 1H, J=8.7, 2.1, H-6â€²).

¹³C-NMR Î´ (CDCl₃, 75 MHz) ppm: 71.0 (CH₂Ph), 71.2 (CH₂Ph), 71.5 (CH₂Ph), 74.3 (CH₂Ph), 76.1 (CH₂Ph), 84.0 (C-6), 97.3 (C-8), 114.0 (C-4-a), 113.8, 115.4 (C-2â€², C-5â€²), 122.4 (C-6â€²), 123.6 (C-1â€²), 127.1, 127.3, 127.5, 128.0, 128.1, 128.2, 128.3, 128.4, 128.5, 128.6, 128.7, 128.9, 129.0, 135.4, 136.8, 137.1 (CH₂Ph) 139.6 (C-3), 148.3 (C-4â€²), 150.9 (C-3â€²), 154.0 (C-8a), 158.6 (C-5), 159.0 (C-2), 160.8 (C-7), 172.6 (C-4).

Example 27Synthesis of 6-(12-(benzyloxy)dodec-1-ynyl)-2-(3,4-dimethoxyphenyl)-3,5,7-trimethoxy-4H-chromen-4-one (8a)

To a solution of 2-(3,4-dimethoxyphenyl)-6-iodo-3,5,7-trimethoxy-4H-chromen-4-one (0.25 g, 0.50 mmol, 1 eq) in DMF (5 mL), piperidine (0.1 mL, 1 mmol, 2 eq), PdCl₂(PPh₃)₂(0.025 g, 0.035 mmol, 0.07 eq), CuI (6.7 mg, 0.035 mmol, 0.07 eq) and 1-((dodec-11-ynyloxy)methyl)benzene (0.21 g, 0.75 mmol, 1.5 eq) were added under argon at room temperature. The reaction was stirred at 80Â° C. overnight. The resulting mixture quenched with 50 g of chilled water and the aqueous phase was extracted with ethyl acetate (3*50 mL). The combined organic layers were washed with brine, dried over magnesium sulfate and concentrated in vacuo. The residue was purified by silica gel column chromatography (heptanes/ethyl acetate: 8/2, 7/3 then 1/1) to afford 0.21 g (66%) of a fatty colourless oil.

Molecular Weight: 642.78 (C₃₉H₄₆O₈).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 1.31 (s large, 12H, H-5â€³ to H-10â€³), 1.41 to 1.70 (m, 4H, H-4â€³,11â€³), 2.53 (t, 2H, J=6.9, H-3â€³), 3.46 (t, 2H, J=6.6, H-12â€³), 3.87 (s, 3H, MeO), 3.97 (s, 9H, MeOÃ—3), 4.04 (s, 3H, MeO), 4.50 (s, 2H, CH₂Ph), 6.68 (s, 1H, H-8), 6.98 (d, 1H, J=9.0, H-5â€²), 7.33 (m, 5H, CH₂Ph), 7.70 (d, 1H, J=2.1, H-2â€²), 7.71 (dd, 1H, J=9.0, 2.1, H-6â€²).

¹³C-NMR Î´ (CDCl₃, 75 MHz) ppm: 20.1 (C-3â€³), 26.3 (C-10â€³), 28.9 to 29.9 (C-4â€³ to C-9â€³, C-11â€³), 56.1 (MeO), 56.3 (MeO), 56.6 (MeO), 60.0 (MeO), 61.9 (MeO), 70.6 (C-12â€³), 71.2 (C-1â€³), 73.0 (CH₂Ph), 95.2 (C-8), 100.1 (C-2â€³), 106.7 (C-4-a), 110.9, 111.3 (C-2â€², C-5â€²), 113.1 (C-6), 121.8 (C-6â€²), 123.3 (C-1â€²), 127.5 (CH₂Ph), 127.7 (CH₂Ph), 128.4 (CH₂Ph), 138.8 (CH₂Ph), 141.2 (C-3), 148.8 (C-4â€²), 151.1 (C-3â€²), 153.3 (C-2), 157.1 (C-8a), 162.6 (C-5), 163.6 (C-7), 173.1 (C-4).

Example 28Synthesis of 2-(3,4-bisbenzyloxyphenyl)-3,7-bisbenzyloxy-6-(12-benzyloxydodec-1-ynyl)-5-methoxy-4H-chromen-4-one (8b)

The compound 8b was prepared by the method of Example 27, but using 2-(3,4-bisbenzyloxyphenyl)-3,7-bisbenzyloxy-6-iodo-5-methoxy-4H-chromen-4-one instead of 2-(3,4-dimethoxyphenyl)-6-iodo-3,5,7-trimethoxy-4H-chromen-4-one.

Molecular Weight: 947.16 (C₆₃H₆₂O₈).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 1.25 (s large, 12H, H-5â€³ to H-10â€³), 1.39 to 1.70 (m, 4H, H-4â€³,11â€³), 2.52 (t, 2H, J=6.9, H-3â€³), 3.46 (t, 2H, J=6.6, H-12â€³), 4.07 (s, 3H, MeO), 4.50 (s, 2H, CH₂Ph), 4.96 (s, 2H, CH₂Ph), 5.06 (s, 2H, CH₂Ph), 5.21 (s, 2H, CH₂Ph), 5.24 (s, 2H, CH₂Ph), 6.65 (s, 1H, H-8), 6.95 (d, 1H, J=8.7, H-5â€²), 7.33 (m, 26H, H-6â€², 5*CH₂Ph), 7.71 (d, 1H, J=2.1, H-2â€²).

Example 29Synthesis of 3,7-bisbenzyloxy-2-(4-benzyloxy-3-methoxyphenyl)-6-(12-benzyloxydodec-1-ynyl)-5-methoxy-4H-chromen-4-one (8c)

The compound 8c was prepared by the method of Example 27, but using 3,7-bisbenzyloxy-2-(4-benzyloxy-3-methoxyphenyl)-6-iodo-5-methoxy-4H-chromen-4-one instead of 2-(3,4-dimethoxyphenyl)-6-iodo-3,5,7-trimethoxy-4H-chromen-4-one.

Molecular Weight: 871.07 (C₅₇H₅₈O₈).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 1.25 (s large, 12H, H-5â€³ to H-10â€³), 1.41 to 1.70 (m, 4H, H-4â€³,11â€³), 2.53 (t, 2H, J=6.9, H-3â€³), 3.46 (t, 2H, J=6.6, H-12â€³), 3.69 (s, 3H, MeO), 4.08 (s, 3H, MeO), 4.50 (s, 2H, CH₂Ph), 5.08 (s, 2H, CH₂Ph), 5.21 (s, 2H, CH₂Ph), 5.23 (s, 2H, CH₂Ph), 6.70 (s, 1H, H-8), 6.93 (d, 1H, J=8.7, H-5â€²), 7.33 (m, 21H, H-6â€², 4*CH₂Ph), 7.64 (d, 1H, J=2.1, H-2â€²).

Example 30Synthesis of 2-(3,4-bisbenzyloxyphenyl)-3,5,7-trisbenzyloxy-6-(12-benzyloxydodec-1-ynyl)-4H-chromen-4-one (8d)

The compound 8d was prepared by the method of Example 27, but using 2-(3,4-bisbenzyloxyphenyl)-3,5,7-trisbenzyloxy-6-iodo-4H-chromen-4-one instead of 2-(3,4-dimethoxyphenyl)-6-iodo-3,5,7-trimethoxy-4H-chromen-4-one.

Molecular Weight: 1023.29 (C₆₉H₆₆O₈).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 1.25 (s large, 12H, H-5â€³ to H-10â€³), 1.39 to 1.70 (m, 4H, H-4â€³,11â€³), 2.47 (t, 2H, J=6.9, H-3â€³), 3.46 (t, 2H, J=6.6, H-12â€³), 4.50 (s, 2H, CH₂Ph), 4.98 (s, 2H, CH₂Ph), 5.06 (s, 2H, CH₂Ph), 5.22 (s, 2H, CH₂Ph), 5.23 (s, 2H, CH₂Ph), 5.24 (s, 2H, CH₂Ph), 6.69 (s, 1H, H-8), 6.95 (d, 1H, J=8.7, H-5â€²), 7.33 (m, 30H, 6*CH₂Ph), 7.71 (d, 1H, J=2.1, H-2â€²), 7.75 (dd, 1H, J=8.7, 2.1, H-6â€²).

Example 31Synthesis of 6-(12-hydroxydodec-1-yl)-2-(3,4-dimethoxyphenyl)-3,5,7-trimethoxy-4H-chromen-4-one (9a)

To a solution of 6-(12-benzyloxydodec-1-ynyl)-2-(3,4-dimethoxyphenyl)-3,5,7-trimethoxy-4H-chromen-4-one (0.20 g, 0.31 mmol, 1 eq) in EtOH/acetone (1/1) (6 mL), was added 10% Pd/C wet Degussaâ„¢ (0.15 g, 38% w/w). The reaction mixture was then stirred under hydrogen atmosphere for 36 h at room temperature. The solution was filtered through Celite and concentrated in vacuo. The residue was purified by silica gel column chromatography (ethyl acetate/heptanes: 1/1 then 4/1) to afford 0.09 g (52%) of a white solid.

Molecular Weight: 556.69 (C₃₂H₄₄O₈).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 1.25 (s large, 16H, H-3â€³ to H-10â€³), 1.51 to 1.60 (m, 4H, H-2â€³,11â€³), 2.66 (t, 2H, J=8.1, H-1â€³), 3.63 (t, 2H, J=6.6, H-12â€³), 3.85 (s, 3H, MeO), 3.91 (s, 3H, MeO), 3.92 (s, 3H, MeO), 3.96 (s, 6H, MeOÃ—2), 6.69 (s, 1H, H-8), 6.98 (d, 1H, J=9.0, H-5â€²), 7.71 (m, 2H, H-2â€², H-6â€²).

¹³C-NMR Î´ (CDCl₃, 75 MHz) ppm: 23.3 (C-1â€³), 25.9 (C-10â€³), 29.6 to 30.0 (C-2â€³ to C-9â€³), 32.9 (C-11â€³), 56.0 (MeO), 56.1 (MeO), 56.2 (MeO), 60.1 (MeO), 62.6 (MeO), 63.2 (C-12â€³), 95.0 (C-8), 110.9, 111.4 (C-2â€², C-5â€²), 112.6 (C-4-a), 121.8 (C-6â€²), 123.2 (C-6), 123.6 (C-1â€²), 141.2 (C-3), 148.8 (C-4â€²), 150.9 (C-3â€²), 153.2 (C-2), 156.7 (C-8a), 157.7 (C-5), 162.4 (C-7), 173.63 (C-4).

Example 32Synthesis of 2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-6-(12-hydroxydodecyl)-5-methoxy-4H-chromen-4-one (9b)

The compound 9b was prepared by the method of Example 31, but using 2-(3,4-bisbenzyloxyphenyl)-3,7-bisbenzyloxy-6-(12-benzyloxydodec-1-ynyl)-5-methoxy-4H-chromen-4-one instead of 6-(12-benzyloxydodec-1-ynyl)-2-(3,4-dimethoxyphenyl)-3,5,7-trimethoxy-4H-chromen-4-one.

Molecular Weight: 500.58 (C₂₈H₃₆O₈).

¹H-NMR Î´ (CD₃OD, 300 MHz) ppm (J in Hz): 1.22 (s large, 16H, H-3â€³ to H-10â€³), 1.50 to 1.60 (m, 4H, H-2â€³,11â€³), 2.64 (t, 2H, J=7.5, H-1â€³), 3.52 (t, 2H, J=6.9, H-12â€³), 3.84 (s, 3H, MeO), 6.69 (s, 1H, H-8), 6.89 (d, 1H, J=8.4, H-5â€²), 7.61 (dd, 1H, J=8.4, 2.1, H-6â€²), 7.70 (d, 1H, J=2.1, H-2â€²).

¹³C-NMR Î´ (CD₃OD, 75 MHz) ppm: 23.7 (C-1â€³), 26.2 (C-10â€³), 29.9 to 30.3 (C-2â€³ to C-9â€³), 32.9 (C-11â€³), 62.7 (MeO), 62.7 (C-12â€³), 98.9 (C-8), 108.9 (C-4-a), 115.0, 115.7 (C-2â€², C-5â€²), 120.8 (C-6), 122.7 (C-6â€²), 123.6 (C-1â€²), 137.5 (C-3), 143.5 (C-4â€²), 145.1 (C-3â€²), 147.3 (C-8a), 156.9 (C-5), 157.7 (C-2), 162.1 (C-7), 172.2 (C-4).

Example 33Synthesis of 3,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-(12-hydroxydodecyl)-5-methoxy-4H-chromen-4-one (9c)

The compound 9c was prepared by the method of Example 31, but using 3,7-bisbenzyloxy-2-(4-benzyloxy-3-methoxyphenyl)-6-(12-benzyloxydodec-1-ynyl)-5-methoxy-4H-chromen-4-one instead of 6-(12-benzyloxydodec-1-ynyl)-2-(3,4-dimethoxyphenyl)-3,5,7-trimethoxy-4H-chromen-4-one.

Molecular Weight: 514.6 (C₂₉H₃₈O₈).

¹H-NMR Î´ (CD₃OD, 300 MHz) ppm (J in Hz): 1.22 (s large, 16H, H-3â€³ to H-10â€³), 1.50 to 1.61 (m, 4H, H-2â€³,11â€³), 2.63 (t, 2H, J=7.5, H-1â€³), 3.52 (t, 2H, J=6.9, H-12â€³), 3.84 (s, 3H, MeO), 3.92 (s, 3H, MeO), 6.67 (s, 1H, H-8), 6.93 (d, 1H, J=8.7, H-5â€²), 7.62 (dd, 1H, J=8.7, 2.1, H-6â€²), 7.78 (d, 1H, J=2.1, H-2â€²).

Example 34Synthesis of 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-6-(12-hydroxydodecyl)-4H-chromen-4-one (9d)

The compound 9d was prepared by the method of Example 31, but using 2-(3,4-bisbenzyloxyphenyl)-3,5,7-trisbenzyloxy-6-(12-benzyloxydodec-1-ynyl)-4H-chromen-4-one instead of 6-(12-benzyloxydodec-1-ynyl)-2-(3,4-dimethoxy phenyl)-3,5,7-trimethoxy-4H-chromen-4-one.

Molecular Weight: 486.55 (C₂₇H₃₄O₈).

¹H-NMR Î´ (CD₃OD, 300 MHz) ppm (J in Hz): 1.25 (s large, 16H, H-3â€³ to H-10â€³), 1.61 to 1.82 (m, 4H, H-2â€³,11â€³), 2.61 (t, 2H, J=7.7, H-1â€³), 3.53 (t, 2H, J=6.8, H-12â€³), 6.41 (s, 1H, H-8), 6.88 (d, 1H, J=8.4, H-5â€²), 7.61 (dd, 1H, J=8.4, J=2.1, H-6â€²), 7.71 (d, 1H, J=2.1, H-2â€²).

¹³C-NMR Î´ (CD₃OD, 75 MHz) ppm: 22.6 (C-1â€³), 26.2 (C-10â€³), 29.2 to 30.0 (C-3â€³ to C-9â€³), 32.9 (C-11â€³), 62.7 (C-12â€³), 93.3 (C-8), 103.7 (C-4-a), 112.8 (C-6), 115.3, 115.6 (C-2â€², C-5â€²), 121.1 (C-6â€²), 123.6 (C-1â€²), 136.1 (C-3), 145.1 (C-3â€²), 146.6 (C-8a), 147.5 (C-4â€²), 155.2 (C-5), 158.2 (C-2), 162.7 (C-7), 176.1 (C-4).

Following the method of Example 31, the following compounds shown in Process B are prepared:

6-(13-Hydroxytridec-1-yl)-2-(3,4-dimethoxyphenyl)-3,5,7-trimethoxy-4H-chromen-4-one
2-(3,4-Dihydroxyphenyl)-3,7-dihydroxy-6-(13-hydroxytridecyl)-5-methoxy-4H-chromen-4-one
3,7-Dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-(13-hydroxytridecyl)-5-methoxy-4H-chromen-4-one
2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-6-(13-hydroxytridecyl)-4H-chromen-4-one

Example Synthesis of Compound 20 of General Formula (I)Example 35Synthesis of 1-(2-hydroxy-4,6-dimethoxyphenyl)ethanone (11)

To a solution of commercial 2â€²,4â€²,6â€²-trihydroxyacetophenone dihydrate (15.0 g, 80.57 mmol, 1 eq) in acetone (170 mL), potassium carbonate (23.4 g, 169.20 mmol, 2.1 eq) and dimethylsulfate (15.6 mL, 165.17 mmol, 2.05 eq) were added under argon at 0Â° C. After vigorous stirring at room temperature for 17 h, the resulting mixture was filtered and the filtrate concentrated in vacuo. The resulting product 5 was recrystallized from ethyl acetate/heptanes (3/97) to give 15.80 g (99%) of a pale yellow solid.

Molecular Weight: 196.20 (C₁₀H₁₂O₄).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 2.61 (s, 3H, CH₃CO), 3.82 (s, 3H, MeO), 3.85 (s, 3H, MeO), 5.91 (d, 1H, J=2.4, H-3â€²), 6.05 (d, 1H, J=2.4, H-5â€²), 14.03 (s, 1H, OH).

Example 36Synthesis of 2-acetyl-3,5-dimethoxyphenyl 3,4-dimethoxybenzoate (12)

3,4-dimethoxybenzoyl chloride (10.55 g, 52.72 mmol, 1.2 eq) was added to a suspension of 1-(2-hydroxy-4,6-dimethoxyphenyl)ethanone (8.62 g, 43.93 mmol, 1 eq) in pyridine (120 mL). The reaction mixture was then stirred at 100Â° C. for 10 mins. After cooling, to this suspension was added ethanol (100 mL) then water (100 mL). This solution was placed at 0Â° C. and following scratching with a glass rod to afford white tiny crystals. The precipitate was filtered, washed with ethanol/water (1/1) (30 mL), dried to give 11.57 g (73%) of a white solid.

Molecular Weight: 360.36 (C₁₉H₂₀O₇).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 2.47 (s, 3H, CH₃CO), 3.82 (s, 3H, MeO), 3.85 (s, 3H, MeO), 3.94 (s, 3H, MeO), 3.95 (s, 3H, MeO), 6.37 (d, 1H, J=2.2, H-3â€²), 6.39 (d, 1H, J=2.2, H-5â€²), 6.92 (d, 1H, J=8.5, H-5â€³), 7.61 (d, 1H, J=2.0, H-2â€³), 7.79 (dd, 1H, J=2.0, J=8.5, H-6â€³).

Following the method of Example 36, the following compounds shown in Process C are prepared:

2-Acetyl-3,5-dimethoxyphenyl 4-methoxybenzoate
2-Acetyl-3,5-dimethoxyphenyl 4-(benzyloxy)-3-methoxybenzoate
2-acetyl-3,5-dimethoxyphenyl 2,4,5-trimethoxybenzoate
2-Acetyl-3,5-dimethoxyphenyl 3,4,5-trimethoxybenzoate

Example 37Synthesis of 1-(3,4-dimethoxyphenyl)-3-(2-hydroxy-4,6-dimethoxyphenyl) propane-1,3-dione (13)

To a suspension of 2-acetyl-3,5-dimethoxyphenyl 3,4-dimethoxybenzoate (10.31 g, 28.61 mmol, 1 eq) in pyridine (50 mL) was added crush hydroxide potassium (4.01 g, 71.52 mmol, 2.5 eq). The reaction mixture was stirred at 100Â° C. for 10 mins. After cooling, this solution was poured into glacial acetic acid (30 mL) then ethanol (100 mL) and water (100 mL) was added. This solution was placed at 0Â° C. and following scratching with a glass rod to afford yellow tiny crystals. The precipitate was filtered, washed with ethanol/water (1/1) (30 mL), dried to give 7.47 g (72%) of a pale yellow solid.

Molecular Weight: 360.36 (C₁₉H₂₀O₇).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): ratio enol/1,3-diketone: (52/48); 1,3-diketone: 3.48 (s, 2H, COCH₂CO), 3.79 (s, 3H, MeO), 3.92 (s, 3H, MeO), 3.95 (s, 6H, MeOÃ—2), 5.82 (d, 1H, J=2.4, H-3â€²), 6.08 (d, 1H, J=2.4, H-5â€²), 6.89 (d, 1H, J=8.1, H-5â€³), 7.53 (d, 1H, J=2.1, H-2â€³), 7.55 (dd, 1H, J=8.1, J=2.1, H-6â€³); enol: 3.80 (s, 3H, MeO), 3.90 (s, 3H, MeO), 3.92 (s, 3H, MeO), 3.95 (s, 3H, MeO), 4.50 (s, 1H, HCâ• C), 5.97 (d, 1H, J=2.4, H-3â€²), 6.09 (d, 1H, J=2.4, H-5â€²), 6.89 (d, 1H, J=8.1, H-5â€³), 7.43 (d, 1H, J=2.1, H-2â€³), 7.50 (dd, 1H, J=8.1, J=2.1, H-6â€³).

Following the method of Example 37, the following compounds shown in Process C are prepared:

1-(2-Hydroxy-4,6-dimethoxyphenyl)-3-(4-methoxyphenyl)propane-1,3-dione
1-(4-Benzyloxy-3-methoxyphenyl)-3-(2-hydroxy-4,6-dimethoxyphenyl) propane-1,3-dione
1-(2-Hydroxy-4,6-dimethoxyphenyl)-3-(2,4,5-trimethoxyphenyl) propane-1,3-dione
1-(2-Hydroxy-4,6-dimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)propane-1,3-dione

Example 38Synthesis of 2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4H-chromen-4-one (14)

A suspension of 1-(3,4-dimethoxyphenyl)-3-(2-hydroxy-4,6-dimethoxyphenyl) propane-1,3-dione (7.40 g, 20.53 mmol, 1 eq) in glacial acetic acid (30 mL) was heated at 100Â° C. To this suspension was added 20 mL of 20% H₂SO₄in acetic acid and the mixture stirred at 100Â° C. for 10 mins. The mixture was poured into 150 g of crushed ice, which produced a pale-yellow gelatinous solid. The solid was collected, allowed to partially dry by drawing air through the funnel, and then partitioned between 300 mL each of CHCl₃and distillated water. The organic layer was separated and washed with 150 mL each of 5% NaHCO₃and saturated brine, dried over MgSO₄, filtered and evaporated to afford a pale yellow solid. Recrystallization from 50 mL of acetone provided 6.05 g (86%) of white needles.

Molecular Weight: 342.34 (C₁₉H₁₈O₆).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 3.86 (s, 3H, MeO), 3.90 (s, 6H, MeOÃ—2), 3.92 (s, 3H, MeO), 6.30 (d, 1H, J=2.1, H-6), 6.48 (d, 1H, J=2.1, H-8), 6.53 (s, 1H, H-3), 6.88 (d, 1H, J=8.6, H-5â€²), 7.24 (d, 1H, J=2.1, H-2â€²), 7.42 (dd, 1H, J=2.1, J=8.6, H-6â€²).

¹³C-NMR Î´ (CDCl₃, 75 MHz) ppm: 55.8 (MeO), 56.1 (MeOÃ—2), 56.4 (MeO), 92.9 (C-6), 96.1 (C-8), 107.8 (C-3), 108.5 (C-2â€²), 109.1 (C-4-a), 111.0 (C-5â€²), 119.5 (C-6â€²), 123.9 (C-1â€²), 149.2 (C-4â€²), 151.7 (C-3â€²), 159.8 (C-8a), 160.6 (C-5), 160.8 (C-2), 163.9 (C-7), 177.6 (C-4).

Following the method of Example 38, the following compounds shown in Process C are prepared:

5,7-Dimethoxy-2-(4-methoxyphenyl)-4H-chromen-4-one
2-(4-Benzyloxy-3-methoxyphenyl)-5,7-dimethoxy-4H-chromen-4-one
5,7-Dimethoxy-2-(2,4,5-trimethoxyphenyl)-4H-chromen-4-one
5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one

Example 39Synthesis of 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxy-4H-chromen-4-one (15)

To a solution of 2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4H-chromen-4-one (6.03 g, 17.61 mmol, 1 eq) in dry dichloromethane (75 mL) at 0Â° C. was added dropwise a solution of boron tribromide 1 M in dichloromethane (17.61 mL, 17.61 mmol, 1 eq). After vigorous stirring at room temperature overnight, the resulting mixture was quenched with ethanol (150 mL) then the solvent was concentrated in vacuo. The yellow solid obtained, was refluxed in ethanol/water (1/1) (300 mL). The solution placed at 0Â° C., filtered, washed with diethyl ether (100 mL) to afford 4.75 g (82%) of a yellow solid.

Molecular Weight: 328.32 (C₁₈H₁₆O₆).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 3.86 (s, 3H, MeO), 3.95 (s, 3H, MeO), 3.96 (s, 3H, MeO), 6.33 (d, 1H, J=2.3, H-6), 6.45 (d, 1H, J=2.3, H-8), 6.54 (s, 1H, H-3), 6.94 (d, 1H, J=8.6, H-5â€²), 7.29 (d, 1H, J=2.1, H-2â€²), 7.48 (dd, 1H, J=2.1, J=8.6, H-6â€²).

¹³C-NMR Î´ (CDCl₃, 75 MHz) ppm: 56.2 (MeOÃ—2), 55.9 (MeO), 92.7 (C-8), 98.2 (C-6), 104.7 (C-3), 105.6 (C-4-a), 108.8 (C-2â€²), 111.2 (C-5â€²), 120.2 (C-6â€²), 123.8 (C-1â€²), 149.4 (C-4â€²), 152.4 (C-3â€²), 157.7 (C-8a), 162.2 (C-5), 164.0 (C-2), 165.5 (C-7), 182.4 (C-4).

Following the method of Example 39, the following compounds shown in Process C are prepared:

5-Hydroxy-7-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one
2-(4-Benzyloxy-3-methoxyphenyl)-5-hydroxy-7-methoxy-4H-chromen-4-one
5-Hydroxy-7-methoxy-2-(2,4,5-trimethoxyphenyl)-4H-chromen-4-one
5-Hydroxy-7-methoxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one

Example 40Synthesis of 2-(3,4-dimethoxyphenyl)-5-hydroxy-6-iodo-7-methoxy-4H-chromen-4-one (16)

To a solution of 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxy-4H-chromen-4-one (4.40 g, 13.40 mmol, 1 eq) in dichloromethane-MeOH (5/2) (210 mL), calcium carbonate (9.39 g, 93.81 mmol, 7 eq) and benzyltrimethyl ammonium chloroiodate (BTMA-ICl₂) (4.66 g, 13.40 mmol, 1 eq) were added under argon at 0Â° C. After vigorous stirring at room temperature overnight, the resulting mixture was quenched with water (150 mL) and extracted three times with dichloromethane (200 mL). The combined organic phases were washed with brine (150 mL), dried over MgSO₄and concentrated in vacuo. The solid obtained was recrystallized from ethanol (150 mL) to afford 5.29 g (87%) of a yellow solid.

Molecular Weight: 454.21 (C₁₈H₁₅IO₆).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 3.96 (s, 3H, MeO), 3.98 (s, 3H, MeO), 3.99 (s, 3H, MeO), 6.51 (s, 1H, H-8), 6.61 (s, 1H, H-3), 6.96 (d, 1H, J=8.6, H-5â€²), 7.31 (d, 1H, J=2.1, H-2â€²), 7.50 (dd, 1H, J=2.1, J=8.6, H-6â€²), 13.79 (s, 1H, OH).

¹³C-NMR Î´ (CDCl₃, 75 MHz) ppm: 56.3 (MeOÃ—2), 57.1 (MeO), 70.1 (C-6), 90.5 (C-8), 104.7 (C-3), 105.7 (C-4-a), 108.9 (C-2â€²), 111.3 (C-5â€²), 120.4 (C-6â€²), 123.5 (C-1â€²), 149.5 (C-4â€²), 152.6 (C-3â€²), 158.2 (C-8a), 161.2 (C-5), 163.4 (C-2), 164.3 (C-7), 181.5 (C-4).

Following the method of Example 40, the following compounds shown in Process C are prepared:

5-Hydroxy-6-iodo-7-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one
2-(4-Benzyloxy-3-methoxyphenyl)-5-hydroxy-6-iodo-7-methoxy-4H-chromen-4-one
5-Hydroxy-6-iodo-7-methoxy-2-(2,4,5-trimethoxyphenyl)-4H-chromen-4-one
5-Hydroxy-6-iodo-7-methoxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one

Example 41Synthesis of 2-(3,4-dimethoxyphenyl)-6-iodo-5,7-dimethoxy-4H-chromen-4-one (17)

To a solution of 2-(3,4-dimethoxyphenyl)-5-hydroxy-6-iodo-7-methoxy-4H-chromen-4-one (0.63 g, 1.38 mmol, 1 eq) in THF (30 mL), potassium carbonate (0.38 g, 2.77 mmol, 2 eq) and dimethylsulfate (0.13 mL, 1.39 mmol, 1 eq) were added under argon at 0Â° C. After vigorous stirring at reflux for 3 h, the resulting mixture was filtered and the filtrate concentrated in vacuo. The yellow solid obtained was purified by silica gel column chromatography (ethyl acetate/heptanes: 1/4, 3/7 then 1/1) to afford 0.43 g (66%) of a white solid.

Molecular Weight: 468.24 (C₁₉H₁₇IO₆).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 3.94 (s, 3H, MeO), 3.96 (s, 3H, MeO), 3.98 (s, 3H, MeO), 4.01 (s, 3H, MeO), 6.63 (s, 1H, H-3), 6.77 (s, 1H, H-8), 6.96 (d, 1H, J=8.5, H-5â€²), 7.30 (d, 1H, J=2.1, H-2â€²), 7.50 (dd, 1H, J=2.1, J=8.5, H-6â€²).

¹³C-NMR Î´ (CDCl₃, 75 MHz) ppm: 56.2 (MeO), 56.3 (MeO), 57.1 (MeO), 62.0 (MeO), 83.2 (C-6), 96.1 (C-8), 107.7 (C-3), 108.8 (C-2â€²), 111.3 (C-5â€²), 119.9 (C-6â€²), 123.8 (C-1â€²), 149.4 (C-4â€²), 152.1 (C-3â€²), 159.8 (C-8a), 160.4 (C-5), 161.4 (C-2), 162.1 (C-7), 176.0 (C-4).

Following the method of Example 41, the following compounds shown in Process C are prepared:

6-Iodo-5,7-dimethoxy-2-(4-methoxyphenyl)-4H-chromen-4-one
2-(4-Benzyloxy-3-methoxyphenyl)-6-iodo-5,7-dimethoxy-4H-chromen-4-one
6-Iodo-5,7-dimethoxy-2-(2,4,5-trimethoxyphenyl)-4H-chromen-4-one
6-Iodo-5,7-dimethoxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one

Example 42Synthesis of 6-(12-benzyloxydodec-1-ynyl)-2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4H-chromen-4-one (18)

To a solution of 2-(3,4-dimethoxyphenyl)-6-iodo-5,7-dimethoxy-4H-chromen-4-one (0.25 g, 0.53 mmol, 1 eq) in DMF (5 mL), piperidine (0.11 mL, 1.07 mmol, 2 eq), (Ph₃P)₂PdCl₂(0.03 g, 0.04 mmol, 0.07 eq), CuI (0.007 g, 0.04 mmol, 0.07 eq) then dodec-11-ynyloxymethylbenzene (0.22 g, 0.80 mmol, 1.5 eq) were added under argon at room temperature. The reaction was stirred at 80Â° C. overnight. The resulting mixture was quenched with chilled water (50 mL) and extracted three times with ethyl acetate (50 mL). The combined organic layers were washed with brine, dried over MgSO₄and concentrated in vacuo. The residue was purified by silica gel column chromatography (ethyl acetate/heptanes: 1/4, 3/7 then 1/1) to afford 0.22 g (66%) of a fatty colourless oil.

Molecular Weight: 612.77 (C₃₈H₄₄O₇).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 1.30 (s large, 12H, H-5â€³ to H-10â€³), 1.42 to 1.70 (m, 4H, H-4â€³,11â€³), 2.53 (t, 2H, J=6.9, H-3â€³), 3.46 (t, 2H, J=6.6, H-12â€³), 3.96 (s, 3H, MeO), 3.97 (s, 3H, MeO), 3.98 (s, 3H, MeO), 4.02 (s, 3H, MeO), 4.50 (s, 2H, CH₂Ph), 6.58 (s, 1H, H-8), 6.72 (s, 1H, H-3), 6.96 (d, 1H, J=8.7, H-5â€²), 7.33 (m, 6H, H-2â€², CH₂Ph), 7.49 (dd, 1H, J=2.4, J=8.7, H-6â€²).

¹³C-NMR Î´ (CDCl₃, 75 MHz) ppm: 20.1 (C-3â€³), 26.3 (C-10â€³), 28.8 to 29.9 (C-4â€³ to C-9â€³, C-11â€³), 56.2 (MeO), 56.2 (MeO), 56.6 (MeO), 61.9 (MeO), 70.6 (C-12â€³), 71.2 (C-1â€³), 73.0 (CH₂Ph), 95.5 (C-8), 100.1 (C-2â€³), 106.9 (C-4-a), 107.9 (C-3), 108.7 (C-2â€²), 111.2 (C-5â€²), 112.8 (C-6), 119.7 (C-6â€²), 124.0 (C-1â€²), 127.5 (CH₂Ph), 127.7 (CH₂Ph), 128.4 (CH₂Ph), 132.3 (CH₂Ph), 149.3 (C-4â€²), 151.9 (C-3â€²), 158.1 (C-8a), 161.1 (C-5), 162.5 (C-2), 163.7 (C-7), 176.5 (C-4).

Following the method of Example 42, the following compounds shown in Process C are prepared:

6-(12-Benzyloxydodec-1-ynyl)-5,7-dimethoxy-2-(4-methoxyphenyl)-4H-chromen-4-one
6-(13-Benzyloxytridec-1-ynyl)-5,7-dimethoxy-2-(4-methoxyphenyl)-4H-chromen-4-one
6-(13-Benzyloxytridec-1-ynyl)-2-(3,4-dimethoxy-phenyl)-5,7-dimethoxy-4H-chromen-4-one
2-(4-Benzyloxy-3-methoxyphenyl)-6-(12-benzyloxydodec-1-ynyl)-5,7-dimethoxy-4H-chromen-4-one
2-(4-Benzyloxy-3-methoxyphenyl)-6-(13-benzyloxytridec-1-ynyl)-5,7-dimethoxy-4H-chromen-4-one
6-(12-Benzyloxydodec-1-ynyl)-5,7-dimethoxy-2-(2,4,5-trimethoxyphenyl)-4H-chromen-4-one
6-(13-Benzyloxytridec-1-ynyl)-5,7-dimethoxy-2-(2,4,5-trimethoxyphenyl)-4H-chromen-4-one
6-(12-Benzyloxydodec-1-ynyl)-5,7-dimethoxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one
6-(13-Benzyloxytridec-1-ynyl)-5,7-dimethoxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one

Example 43Synthesis of 6-(12-hydroxydodec-1-yl)-2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4H-chromen-4-one (19)

To a solution of 6-(12-benzyloxy-dodec-1-ynyl)-2-(3,4-dimethoxy-phenyl)-5,7-dimethoxy-4H-chromen-4-one (0.22 g, 0.35 mmol, 1 eq) in EtOH/acetone/water (10/10/1) (10.5 mL), was added 10% Pd/C (0.3 g, 136% w/w). The reaction mixture was then stirred under hydrogen atmosphere for 36 h at room temperature. The solution was filtered through Celite and concentrated in vacuo. The residue was purified by silica gel column chromatography (ethyl acetate/heptanes: 1/1 then 4/1) to afford 0.10 g (53%) of a white solid.

Molecular Weight: 526.66 (C₃₁H₄₂O₇).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 1.25 (s large, 16H, H-3â€³ to H-10â€³), 1.52 to 1.60 (m, 4H, H-2â€³,11â€³), 2.66 (t, 2H, J=7.8, H-1â€³), 3.63 (t, 2H, J=6.6, H-12â€³), 3.89 (s, 3H, MeO), 3.93 (s, 3H, MeO), 3.95 (s, 3H, MeO), 3.98 (s, 3H, MeO), 6.60 (s, 1H, H-8), 6.74 (s, 1H, H-3), 6.97 (d, 1H, J=8.7, H-5â€²), 7.33 (d, 1H, J=2.4, H-2â€²), 7.50 (dd, 1H, J=2.4, J=8.7, H-6â€²).

¹³C-NMR Î´ (CDCl₃, 75 MHz) ppm: 23.3 (C-1â€³), 25.9 (C-10â€³), 29.6 to 30.0 (C-2â€³ to C-9â€³), 33.0 (C-11â€³), 56.1 (MeO), 56.2 (MeO), 56.3 (MeO), 62.6 (MeO), 63.2 (C-12â€³), 95.3 (C-8), 107.9 (C-3), 108.8 (C-2â€²), 111.2 (C-5â€²), 112.3 (C-4-a), 119.7 (C-6â€²), 123.5 (C-6), 124.4 (C-1â€²), 149.4 (C-4â€²), 151.8 (C-3â€²), 157.7 (C-8a), 157.8 (C-5), 161.0 (C-2), 162.4 (C-7), 177.2 (C-4).

Following the method of Example 43, the following compounds shown in Process C are prepared:

6-(12-Hydroxydodecyl)-5,7-dimethoxy-2-(4-methoxyphenyl)-4H-chromen-4-one
6-(13-Hydroxytridecyl)-5,7-dimethoxy-2-(4-methoxyphenyl)-4H-chromen-4-one
6-(13-Hydroxytridec-1-yl)-2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4H-chromen-4-one
2-(4-Hydroxy-3-methoxyphenyl)-6-(12-hydroxydodecyl)-5,7-dimethoxy-4H-chromen-4-one
2-(4-Hydroxy-3-methoxyphenyl)-6-(13-hydroxytridecyl)-5,7-dimethoxy-4H-chromen-4-one
6-(12-Hydroxydodecyl)-5,7-dimethoxy-2-(2,4,5-trimethoxyphenyl)-4H-chromen-4-one
6-(13-Hydroxytridecyl)-5,7-dimethoxy-2-(2,4,5-trimethoxyphenyl)-4H-chromen-4-one
6-(12-Hydroxydodecyl)-5,7-dimethoxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one
6-(13-Hydroxytridecyl)-5,7-dimethoxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one

Example 44Synthesis of 2-(3,4-dihydroxyphenyl)-5-hydroxy-6-(12-hydroxydodecyl)-7-methoxy-4H-chromen-4-one (20)

To a solution of 6-(12-hydroxydodec-1-yl)-2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4H-chromen-4-one (0.02 g, 0.04 mmol, 1 eq) in dichloromethane (5 mL) at 0Â° C., was added boron tribromide in dichloromethane (1.0 M, 0.5 mL, 0.49 mmol, 12 eq). The mixture was warmed to room temperature and then stirred for 22 h. The reaction mixture was then cooled to 0Â° C. and methanol (5 mL) was added. The reaction mixture was heated to reflux for 2 h, and then concentrated in vacuo to give a yellow solid. Water (30 mL) was added and the aqueous phase was extracted with ethyl acetate (3*30 mL). The combined organic layers were washed with brine, dried over magnesium sulfate and concentrated in vacuo. The residue was precipitated from aqueous ethanol to give 0.01 g (50%) of a pale-yellow solid.

Molecular Weight: 484.59 (C₂₈H₃₆O₇).

¹H-NMR Î´ (acetone-d6, 300 MHz) ppm (J in Hz): 1.30 (s large, 16H, H-3â€³ to H-10â€³), 1.56 to 1.83 (m, 4H, H-2â€³,11â€³), 2.64 (t, 2H, J=7.2, H-1â€³), 3.48 (t, 2H, J=6.9, H-12â€³), 3.99 (s, 3H, MeO), 6.62 (s, 1H, H-3), 6.77 (s, 1H, H-8), 7.01 (d, 1H, J=8.7, H-5â€²), 7.50 (m, 2H, H-2â€², 6â€²).

Following the method of Example 44, the following compounds shown in Process C are prepared:

5-Hydroxy-6-(12-hydroxydodecyl)-2-(4-hydroxyphenyl)-7-methoxy-4H-chromen-4-one
5-Hydroxy-2-(4-hydroxyphenyl)-6-(13-hydroxytridecyl)-7-methoxy-4H-chromen-4-one
2-(3,4-Dihydroxyphenyl)-5-hydroxy-6-(13-hydroxytridecyl)-7-methoxy-4H-chromen-4-one
5-Hydroxy-6-(12-hydroxydodecyl)-7-methoxy-2-(2,4,5-trihydroxyphenyl)-4H-chromen-4-one
5-Hydroxy-6-(13-hydroxytridecyl)-7-methoxy-2-(2,4,5-trihydroxyphenyl)-4H-chromen-4-one
5-Hydroxy-6-(12-hydroxydodecyl)-7-methoxy-2-(3,4,5-trihydroxyphenyl)-4H-chromen-4-one
5-Hydroxy-6-(13-hydroxytridecyl)-7-methoxy-2-(3,4,5-trihydroxyphenyl)-4H-chromen-4-one
5,7-Dihydroxy-6-(12-hydroxydodecyl)-2-(4-hydroxyphenyl)-4H-chromen-4-one
5,7-Dihydroxy-2-(4-hydroxyphenyl)-6-(13-hydroxytridecyl)-4H-chromen-4-one
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-6-(12-hydroxydodecyl)-4H-chromen-4-one
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-6-(13-hydroxytridecyl)-4H-chromen-4-one
5,7-Dihydroxy-6-(12-hydroxydodecyl)-2-(2,4,5-trihydroxyphenyl)-4H-chromen-4-one
5,7-Dihydroxy-6-(13-hydroxytridecyl)-2-(2,4,5-trihydroxyphenyl)-4H-chromen-4-one
5,7-Dihydroxy-6-(12-hydroxydodecyl)-2-(3,4,5-trihydroxyphenyl)-4H-chromen-4-one
5,7-Dihydroxy-6-(13-hydroxytridecyl)-2-(3,4,5-trihydroxyphenyl)-4H-chromen-4-one

Example Synthesis of Compound of General Formula (II)Example 45Synthesis of 2-(10-benzyloxydecyl)-7-(3,4-dimethoxyphenyl)-4,8-dimethoxy-9H-furo[2,3-f]chromen-9-one (21)

To a solution of 2-(3,4-dimethoxyphenyl)-6-iodo-3,5,7-trimethoxy-4H-chromen-4-one (0.10 g, 0.20 mmol, 1 eq) in piperidine (5 mL), PdCl₂P(Ph₃)₂(0.01 g, 0.014 mmol, 0.07 eq) and CuI (0.003 g, 0.014 mmol, 0.07 eq) were added under argon, followed by dodec-11-ynyloxymethylbenzene (0.08 g, 0.30 mmol, 1.5 eq). The reaction was stirred at 80Â° C. overnight. The resulting mixture was quenched with crushed ice, extracted three times with ethyl acetate (50 mL). The combined organic phases were washed with brine, dried over MgSO₄and concentrated in vacuo. The residue was purified by silica gel column chromatography (ethyl acetate/heptanes: 1/4, 3/7 then 1/1) to afford 0.08 g (62%) of a pale yellow solid.

Molecular Weight: 628.75 (C₃₈H₄₄O₈).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 1.30 (s large, 8H, H-5â€³ to H-8â€³), 1.41 to 1.70 (m, 4H, H-4â€³,9â€³), 2.90 (t, 2H, J=7.5, H-3â€³), 3.46 (t, J=6.6, H-10â€³)), 3.87 (s, 3H, MeO), 3.97 (s, 9H, MeOÃ—3), 4.04 (s, 3H, MeO), 4.50 (s, 2H, CH₂Ph), 6.68 (s, 1H, H-5), 6.74 (s, 1H, H-2), 6.98 (d, 1H, J=9.0, H-5â€²), 7.33 (m, 5H, CH₂Ph), 7.70 (d, 1H, J=2.1, H-2â€²), 7.71 (dd, 1H, J=2.1, J=9.0, H-6â€²).

Following the method of Example 45, the following compounds shown in Process D are prepared:

2-(11-Benzyloxyundecyl)-7-(3,4-dimethoxyphenyl)-4,8-dimethoxy-9H-furo[2,3-f]chromen-9-one
2-(12-Benzyloxydodecyl)-7-(3,4-dimethoxyphenyl)-4,8-dimethoxy-9H-furo[2,3-f]chromen-9-one
2-(13-Benzyloxytridecyl)-7-(3,4-dimethoxyphenyl)-4,8-dimethoxy-9H-furo[2,3-f]chromen-9-one
2-(14-Benzyloxytetradecyl)-7-(3,4-dimethoxyphenyl)-4,8-dimethoxy-9H-furo[2,3-f]chromen-9-one

Example 46Synthesis of 2-(10-benzyloxydecyl)-7-(3,4-dimethoxyphenyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one (25)

To a solution of 2-(3,4-dimethoxyphenyl)-6-iodo-5,7-dimethoxy-4H-chromen-4-one (0.41 g, 0.89 mmol, 1 eq) in piperidine (5 mL), PdCl₂P(Ph₃)₂(0.044 g, 0.062 mmol, 0.07 eq) and CuI (0.012 g, 0.062 mmol, 0.07 eq) were added followed by dodec-11-ynyloxymethylbenzene (0.36 g, 1.33 mmol, 1.5 eq). The reaction was stirred at 100Â° C. overnight. The resulting mixture was quenched with chilled ice water, extracted three times with ethyl acetate (50 mL). The combined organic phases were washed with brine, dried over MgSO₄. and concentrated in vacuo. The residue was purified by silica gel column chromatography (ethyl acetate/heptanes: 1/4, 3/7 then 1/1) to afford 0.55 g (44%) of a pale yellow solid.

Molecular Weight: 598.73 (C₃₇H₄₂O₇).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 1.30 (s large, 8H, H-5â€³ to H-8â€³), 1.41 to 1.70 (m, 4H, H-4â€³,9â€³), 2.90 (t, 2H, J=7.5, H-3â€³), 3.46 (t, J=6.6, H-10â€³), 3.94 (s, 3H, MeO), 3.97 (s, 3H, MeO), 4.01 (s, 3H, MeO), 4.48 (s, 2H, CH₂Ph), 6.49 (s, 1H, H-5), 6.77 (s, 1H, H-8), 6.79 (s, 1H, H-3), 6.93 (d, 1H, J=9.0, H-5â€²), 7.33 (m, 5H, CH₂Ph), 7.70 (d, 1H, J=2.1, H-2â€²), 7.71 (dd, 1H, J=2.1, J=9.0, H-6â€²).

Following the method of Example 46, the following compounds shown in Process E are prepared:

2-(11-Benzyloxyundecyl)-7-(3,4-dimethoxyphenyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one
2-(12-Benzyloxydodecyl)-7-(3,4-dimethoxyphenyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one
2-(13-Benzyloxytridecyl)-7-(3,4-dimethoxyphenyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one
2-(14-Benzyloxytetradecyl)-7-(3,4-dimethoxyphenyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one
7-(4-Benzyloxy-3-methoxyphenyl)-2-(10-benzyloxydecyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one
7-(4-Benzyloxy-3-methoxyphenyl)-2-(11-benzyloxyundecyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one
7-(4-Benzyloxy-3-methoxyphenyl)-2-(12-benzyloxydodecyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one
7-(4-Benzyloxy-3-methoxyphenyl)-2-(13-benzyloxytridecyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one
7-(4-Benzyloxy-3-methoxyphenyl)-2-(14-benzyloxytetradecyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one

Example 47Synthesis of 7-(3,4-dimethoxyphenyl)-2-(10-hydroxydecyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one (26)

To a solution of 2-(10-benzyloxydecyl)-7-(3,4-dimethoxyphenyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one (0.23 g, 0.38 mmol, 1 eq) in EtOH-cyclohexene (4/1) (16 mL) was added 15-20% Pd(OH)₂/C (0.07 g, 30% w/w). After vigorous stirring at reflux overnight, the solution was filtered through Celite and concentrated in vacuo. The residue was purified by silica gel column chromatography (ethyl acetate/heptanes: 1/4, 3/7 then 1/1) to afford 0.10 g (52%) of a white solid.

Molecular Weight: 508.60 (C₃₀H₃₆O₇).

¹H-NMR Î´ (CDCl₃, 300 MHz) ppm (J in Hz): 1.30 (s large, 12H, H-3â€³ to H-8â€³), 1.52 to 1.70 (m, 4H, H-2â€³,9â€³), 2.89 (t, 2H, J=7.5, H-1â€³), 3.64 (t, J=6.6, H-10â€³), 3.96 (s, 3H, MeO), 3.99 (s, 3H, MeO), 4.04 (s, 3H, MeO), 6.52 (s, 1H, H-5), 6.77 (s, 1H, H-8), 6.79 (s, 1H, H-3), 6.98 (d, 1H, J=9.0, H-5â€²), 7.37 (d, 1H, J=2.1, H-2â€²), 7.55 (dd, 1H, J=2.1, J=9.0, H-6â€²).

Following the method of Example 47, the following compounds shown in Process E are prepared:

7-(3,4-Dimethoxyphenyl)-2-(11-hydroxyundecyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one
7-(3,4-Dimethoxyphenyl)-2-(12-hydroxydodecyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one
7-(3,4-Dimethoxyphenyl)-2-(13-hydroxytridecyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one
7-(3,4-Dimethoxyphenyl)-2-(14-hydroxytetradecyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one
7-(4-Hydroxy-3-methoxyphenyl)-2-(10-hydroxydecyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one
7-(4-Hydroxy-3-methoxyphenyl)-2-(11-hydroxyundecyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one
7-(4-Hydroxy-3-methoxyphenyl)-2-(12-hydroxydodecyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one
7-(4-Hydroxy-3-methoxyphenyl)-2-(13-hydroxytridecyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one
7-(4-Hydroxy-3-methoxyphenyl)-2-(14-hydroxytetradecyl)-4-methoxy-9H-furo[2,3-f]chromen-9-one

Biological AssaysInhibition of the Activation of Microglial Cells

The experiments that were carried out concern the ability of these molecules to inhibit the liberation of nitrites and Tumor Necrosis Factor-alpha (TNF-Î±) in the activated microglia. NO and TNF-Î± production are two common parameters related to inflammatory processes.

Example 48Nitrite Quantification by Griess Method

To evaluate the nitrite production, microglial cells were cultured in DMEM-F12 medium containing 2% heat-inactivated FBS in 48 well plates at a density of 20000 cells per well. Peripheral wells of the plate were discarded. After 72 h, 400 Î¼L out of 500 Î¼L medium was replaced by 350 Î¼L fresh medium. The cells were treated with each molecule to be tested by adding 50 Î¼L of a 10Ã— solution to obtain a final concentration ranging from 0.1 Î¼M to 1 Î¼M. Each treatment was done in duplicates. 10 ng/ml LPS was added after 1 hour and the medium was collected after 48 h and stored at âˆ’20Â° C. until analysis. To quantify the NO production, nitrites were measured using Griess reactive (sulfanilamide 1%, Sigma; N-(1-Naphthyl)ethylenediamine 0.1%, Sigma; phosphoric acid 2.5%). Briefly, 100 Î¼L of medium was placed into a 96 well plate and 100 Î¼L of Griess reactive was added. After 10 nm incubation at RT in the dark, the OD was measured at 550 nm.

The ability of each test compound to decrease the production of nitrite is presented as a percent decrease in OD with respect to the absorbance of the control condition i.e. 0.1% DMSO. Examples of the results are shown in Table 1.

% of NO/Test compound at 1 Î¼Mcontrol [Image Omitted]

87.7 Â± 7.7 [Image Omitted]

89.0 Â± 7.6 [Image Omitted]

72.0 Â± 3.0 [Image Omitted]

61.1 Â± 3.1 [Image Omitted]

59.8 Â± 2.1 [Image Omitted]

77.5 Â± 3.9 [Image Omitted]

84.5 Â± 5.9 [Image Omitted]

86.3 Â± 4.2 [Image Omitted]

95.3 Â± 4.8 [Image Omitted]

103.3 Â± 4.0â€‚ [Image Omitted]

90.3 Â± 3.9 [Image Omitted]

98.2 Â± 1.0 [Image Omitted]

99.6 Â± 1.5 [Image Omitted]

108.7 Â± 10.5 [Image Omitted]

81.6 Â± 4.0 [Image Omitted]

82.4 Â± 6.3 [Image Omitted]

111.7 Â± 1.7â€‚ [Image Omitted]

91.5 Â± 2.6 [Image Omitted]

97.6 Â± 6.9 [Image Omitted]

96.6 Â± 4.2 [Image Omitted]

101.6 Â± 3.8â€‚ [Image Omitted]

85.0 Â± 0.9 [Image Omitted]

112.0 Â± 8.0â€‚ [Image Omitted]

111.7 Â± 9.2â€‚

The compound of Example 13 was revealed to be the most potent from the structure-activity relationship in this screening assay. Moreover, the presence of a side chain Ï‰-alkanol on position 6 of the flavonol moiety was found to be important. There was a loss of activity when only an alkyl side chain was present. The length of the side chain w-alkanol was also found to be important for this anti-inflammatory activity and the best one was that with thirteen carbon atoms.

Example 49TNF-Î± Quantification by ELISA

To quantify the presence of TNF-Î± in the supernatant of microglial cells, the medium was collected 48 h after treatment as described above. The ELISA 96 well plate was incubated overnight with the capture antibody (0.8 Î¼g/ml, R&D). 10 Î¼l of each sample was diluted with 90 Î¼L of incubation buffer (PBS-BSA 1%, pH 7.2-7.4) and the plate was incubated for 2 h at RT. The plate was washed 3 times and incubated for 2 h with the detection antibody (150 ng/mL, R&D). After another washing step, the plate was incubated with streptavidine-HRP (20 nm) and the peroxydase revealed with the R&D detection kit. After 20 nm incubation at RT, the reaction was stopped with an equal volume of a 10% solution of sulfuric acid and the OD was measured at 450 nm.

The ability of each test compound to decrease the production of TNF-Î± is presented as a percent decrease in OD with respect to the absorbance of the control condition i.e. 0.1% DMSO. Examples of the results are shown in Table 2.

% of TNF-Î±/Test compound at 1 Î¼Mcontrol [Image Omitted]

69.2 Â± 3.9 [Image Omitted]

64.7 Â± 2.8 [Image Omitted]

77.2 Â± 9.4 [Image Omitted]

â€‚75.4 Â± 17.2 [Image Omitted]

91.2 Â± 7.9

As for Example 49, the best compounds inhibiting the release of pro-inflammatory cytokines like TNF-Î± were those of Example 13. Comparison of the test results for the compounds of the invention with that obtained for the 6-alkyl chromenone demonstrates that the presence of the Ï‰-alkanol side chain is important for the activity.

Example 50RNA Extraction, Reverse Transcription and Real-Time PCR

To evaluate mRNA expression levels, total RNA was extracted using invisorb

RNA extraction kit (Invitek). The concentration was determined by reading the OD at 260 nm. Retro-transcription was performed using Im-Prom-IIâ„¢ Reverse Transcription System (Promega) with 1 Î¼g of total RNA.

The reaction mixture contained 12.5 Î¼l 2Ã— SYBR Green mastermix (Biorad), 1 Î¼l cDNA, 1 Î¼l of each primer (12.5 Î¼M) and 9.5 Î¼l of H₂O. The real-time PCR amplification was controlled by the MyIQ5 system (Biorad). After activation of the enzyme at 94Â° C. for 3 nm, the amplification was done by cycling 40 times between a denaturation step at 94Â° C. and the annealing step at 54.5Â° C. Gene expression was analyzed using the provided software and normalized to beta-actin expression.

The results showed that compounds according to the invention are able to significantly down-regulate iNOS, pro-inflammatory cytokines and SOCS3 gene expression in microglial cells as shown in FIG. 1.

[Image Omitted]

FIG. 1: Compound B inhibits microglial activation in vitro. Real-time PCR gene expression analysis in microglial cell line. RNA extraction was performed after 3 hours of incubation with compound B (1 Î¼M), with or without LPS activation (0.01 Î¼g/ml). Expression of transcripts in treated microglial cells are significantly decreased. Î²-actin was used as an internal control.

Cell Differentiation

The experiments that were carried out concern the ability of these molecules to induce the differentiation of NSCs towards the neuronal lineage and the differentiation of human neuroblastoma cell line into neurons by inhibiting the Notch signaling pathway. Moreover, if these compounds modulate cell differentiation through inhibition of the Notch signaling pathway, they can be useful for the treatment of cancer where the Notch signaling is up-regulated.

Example 51Differentiation of NSCs to neurons

The NSCs derived neurospheres culture was established from embryonic mouse cortices. Neurospheres were maintained in Neurobasal-A medium supplemented with B27 w/o retinoic acid, 2 mM Glutamine, 20 ng/ml EGF and antibiotics. The neurospheres were dissociated using a non enzymatic dissociation medium (Invitrogen) and the single cell suspension added to a flask with fresh medium. To differentiate the neurospheres into neurons, oligodendrocytes and astrocytes, 3 to 4 days old neurospheres were cultured in poly-ornithine treated 35 mm dishes. Cells were treated with test compounds (1 Î¼M) or 0.1% DMSO vehicle as control in Neurobasal-A medium supplemented with B27 w/o retinoic acid, 2 mM Glutamine, antibiotics, 2 ng/ml EGF and 0.5% of heat inactivated FBS. RNA extraction was performed after 24 hours of incubation and gene expression was analyzed by real-time PCR.

The primer pairs for mouse Mash1, Hes5 and beta-actin were designed with the beacon designer software (premierbiosoft).

GeneForwardReverse(Accessionâ€ƒNo.)PrimerPrimerBeta-actin5â€²-AGGGAAATCGTGC5â€²-GGAGGAAGAGG(NM_007393)GTGACATCAAAGAG-3â€²ATGCGGCAGTGG-3â€² Mash15â€²-AAGATGAGC5â€²-AGTCGTTGG(NM_008553)AAGGTGGAG-3â€²AGTAGTTGG-3â€² Hes55â€²-CGGTGGTGG5â€²-CTTGGAGTT(NM_010419)AGAAGATGC-3â€²GGGCTGGTG-3â€²

Notch signaling controls a wide variety of cell fate decisions, including the differentiation of NSCs during neurogenesis [Artavanis-Tsakonas, S. et al., Science, 1999, 284, 770]. Hence, compounds inhibiting the Notch signaling pathway would increase the production of neurons from NSCs. Notch activation induces the expression of the downstream effectors Hes1 and Hes5, which down-regulate gene transcription of b-HLH transcription factors such as Mash1. Therefore, we tested whether compounds of general Formula (I) and (II) could modulate Notch signalling in NSCs. Interestingly, Hes5 expression, assessed by real-time PCR, were decreased in NSCs cultures treated with example compounds A and B at 1 Î¼M (FIG. 2). Inhibition of the Hes5 gene expression in the treated cultures was further correlated with an increase of Mash1 transcripts, a b-HLH factor involved in neuronal specification and differentiation [Ito, H. et al., J Neurosci Res. 2003, 71, 648]. Altogether, these results provide compiling evidences indicating that compounds according to the invention promote NSCs differentiation, through the inhibition of Notch signaling.

[Image Omitted]

FIG. 2: Compound A and B induces NSCs differentiation into neurons. Real-time PCR gene expression analysis in neurospheres. RNA extraction was performed after 6 hours of incubation with compound A and B (1 Î¼M) or with 0.1% DMSO vehicle as control. Expression of Mash1 transcripts in treated neurospheres are significantly increased with a decrease of Hes5 gene expression. Î²-actin was used as an internal control.

Example 52Differentiation of Human Neuroblastoma Cell Line SH-SY5Y into Neurons

SH-SY5Y cells were cultured in 6 well plates at a density of 500000 cells per well and was maintained in DMEM with 10% FBS. After 24 h, the cells were treated with test compounds (1 Î¼M) or 0.1% DMSO vehicle as control. RNA extraction was performed after 24 hours of incubation and gene expression was analyzed by real-time PCR.

The specific primer pairs directed against human Hash1, Notch1 and beta-actin are from the online database qprimerdepot (http://primerdepot.nci.nih.gov/).

Geneforwardreverse(Accessionâ€ƒNo.)primerprimerBeta-actin5â€²-GCACAGAG5â€²-CCTTGCAC(NM_001101)CCTCGCCTT-3â€²ATGCCGGAG-3â€² HASH15â€²-GGAGCTTCTC5â€²-CTAAAGATGC(NM_004316)GACTTCACCA-3â€²AGGTTGTGCG-3â€² Notch15â€²-AGTGTGAAG5â€²-ATAGTCTGC(NM_017617)CGGCCAATG-3â€²CACGCCTCTG-3â€²

The human homolog of Mash1, Hash1, is expressed in neuroblastoma cell lines. However, Hash1 transcripts became downregulated in several cell lines during induced differentiation [Ichimiya, S. et al., Med Pediatr Oncol. 2001, 36,132]. Therefore, we tested whether compounds of general Formula (I) and (II) could induce the differentiation of SH-SY5Y neuroblastoma cell line by modulating the gene expression of Hash1 and Notch1. Fortunately, cultures treated with example compounds A and B at 1 Î¼M (FIG. 3) proved to inhibit the expression of both Hash1 and Notch1 transcripts correlating with the differentiation of neuroblastoma cell lines into a neuronal phenotype.

[Image Omitted]

FIG. 3: Compound A and B induces neuroblastoma differentiation into a neuronal phenotype. Real-time PCR gene expression analysis in SH-SY5Y neuroblastoma cell line. RNA extraction was performed after 24 hours of incubation with compound A and B (1 Î¼M) or with 0.1% DMSO vehicle as control. Expression of Hash1 and Notch1 transcripts in treated SH-SY5Y cell line are significantly decreased. Î²-actin was used as an internal control.

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